JP2004524002A - Transgenic mice containing targeted gene disruption - Google Patents

Abstract

The present invention relates to transgenic animals and methods and methods for characterizing gene function. Specifically, the present invention provides a transgenic cell comprising a disruption in a target gene. The transgenic cells of the present invention include any cells capable of performing homologous recombination. Preferably, the cells of the invention are stem cells, and more preferably, embryonic stem (ES) cells, and most preferably, mouse ES cells. According to one embodiment, transgenic cells are produced by introducing a targeting construct into a stem cell and generating a homologous recombinant that results in a mutation in the target gene. In another embodiment, the transgenic cells are from a transgenic animal described below. Cells from a transgenic animal include cells isolated from or present in tissues or organs, and any cell line or any progeny thereof.

Description

発現。総ＲＮＡを、成体Ｃ５７Ｂ１／６野生型マウス由来の器官または組織から単離した。ＲＮＡをＤＮａｓｅＩ処理し、そしてランダムプライマーを使用して逆転写した。得られたｃＤＮＡを、転写されないゲノムマウスＤＮＡに特異的なプライマーを使用して、ゲノムの混入の非存在について調べた。ｃＤＮＡを、ＨＰＲＴプライマーを使用して濃度について平均化した。ＲＮＡ転写物は、大脳、皮質、皮質下領域、小脳、脳幹、嗅球、脊髄、眼、ハルダー腺、心臓、肺、肝臓、膵臓、腎臓、脾臓、胸腺、リンパ節、骨髄、皮膚、膀胱、下垂体、副腎、唾液腺、骨格筋、舌、胃、小腸、大腸、盲腸、精巣、精巣上体、精嚢、凝固腺（ｃｏａｇｕｌａｔｉｎｇ ｇｌａｎｄ）、前立腺、卵巣、子宮および脂肪組織（ｗｈｉｔｅ ｆａｔ）において検出可能であった。ＲＮＡ転写物は、胆嚢において検出可能ではなかった。
【０２０３】
（挙動）
挙動研究について、ホモ接合マウスを以下の通りに作製した：
上記の標的化構築物を、１２９／ＳｖＥｖマウス亜系由来のＥＳ細胞に導入して、キメラマウスを作製する。Ｆ１Ｎ０マウスを、Ｃ５７ＢＬ／６の雌と交配することによって作製した。Ｆ２Ｎ０ホモ接合変異マウスを、Ｆ１ヘテロ接合体の雄と雌を異種交配することによって作製した。Ｆ１Ｎ０ヘテロ接合体を、Ｃ５７ＢＬ／６マウスに戻し交配して、Ｆ１Ｎ１ヘテロ接合体を作製した。Ｆ２Ｎ１ホモ接合マウスを、Ｆ１Ｎ１ヘテロ接合体の雄と雌を異種交配することによって作製した。
【０２０４】
ホモ接合マウスは、以下の挙動表現型を示した：
ホモ接合マウスは、特徴的な発作様応答（発作に対して増加した感受性を示す）を誘発するために、有意により少ない用量のメトラゾール（ｍｅｔｒａｚｏｌ）を必要とした。ホモ接合変異体はまた、９０ｄＢの前パルスを有する有意に減少したＰＰＩ（分裂病患者において観察されるのと同様な刺激処理欠損を示す）を示した。
【０２０５】
（実施例２：５−ＨＴ５Ａ遺伝子破壊を含むマウスの作製および分析）
５−ＨＴ５Ａ遺伝子の役割を調査するために、５−ＨＴ５Ａ遺伝子中の破壊を、相同組換えによって生成した。詳細には、５−ＨＴ５Ａ遺伝子中に破壊を含むトランスジェニックマウスを作製した。より詳細には、図５Ａ〜図５Ｂに示されるように、５−ＨＴ５Ａ遺伝子（詳細には、配列番号５を含む）を破壊または改変する能力を有する５−ＨＴ５Ａ特異的標的化構築物を、本明細書中で配列番号７または配列番号８と同定されるオリゴヌクレオチド配列を、この構築物において標的化アーム（相同配列）として使用して作製した。
【０２０６】
この標的化構築物を、１２９／Ｓｖ−＋Ｐ＋Ｍｇｆ−ＳＬＪ／Ｊマウス亜系に由来するＥＳ細胞に導入し、キメラマウスを作製した。Ｆ１マウスを、Ｃ５７ＢＬ／６の雌と交配することによって作製し、そしてＦ２ホモ接合変異マウスを、Ｆ１ヘテロ接合体の雄と雌を異種交配することによって作製した。
【０２０７】
５−ＨＴ５Ａ遺伝子中に破壊を含むトランスジェニックマウスを、表現型変化および発現パターンについて分析した。
【０２０８】
発現。ＬａｃＺ（β−ガラクトシダーゼ）発現は、脳および食道において検出可能であった。脳において、染色は、プルキンエ細胞を有する小脳および発現を示す顆粒層の核に限定された。ホールマウント（ｗｈｏｌｅｍｏｕｎｔ）染色において、ｌａｃＺ発現は、小脳のみで検出可能であった。小脳の冠状切片は、Ｘ−Ｇａｌシグナルが、プルキンエ細胞層に限定されたことを明らかにした。さらに、プルキンエ細胞に隣接した別の核は、染色を示した。強いＸ−ｇａｌシグナルがまた、扁平上皮細胞に存在した。
【０２０９】
（実施例３：コーディン（ｃｈｏｒｄｉｎ）遺伝子破壊を含むマウスの作製および分析）
コーディンの役割を調査するために、コーディン遺伝子中の破壊を、相同組換えにより作製した。詳細には、コーディン遺伝子中に破壊を含むトランスジェニックマウスを作製した。より詳細には、図５Ａ〜図５Ｂに示されるように、コーディン遺伝子（詳細には、配列番号９を含む）を破壊または改変する能力を有するコーディン特異的標的化構築物を、本明細書中で配列番号１１または配列番号１２と同定されるオリゴヌクレオチド配列を、この構築物において標的化アーム（相同配列）として使用して作製した。
【０２１０】
この標的化構築物を、１２９／Ｓｖ−＋Ｐ＋Ｍｇｆ−ＳＬＪ／Ｊマウス亜系に由来するＥＳ細胞に導入し、キメラマウスを作製した。Ｆ１マウスを、Ｃ５７ＢＬ／６の雌と交配することによって作製し、そしてＦ２ホモ接合変異マウスを、Ｆ１ヘテロ接合体の雄と雌を異種交配することによって作製した。
【０２１１】
コーディン遺伝子中に破壊を含むトランスジェニックマウスを、表現型の変化および発現パターンについて分析した。コーディン遺伝子中の破壊に関連する表現型を決定した。ホモ接合マウスは、以下の表現型の少なくとも１つを示した：異常な痛覚、疼痛および不安に対する減少した応答。
【０２１２】
発現。総ＲＮＡを、成体Ｃ５７Ｂ１／６野生型マウス由来の器官または組織から単離した。ＲＮＡをＤＮａｓｅＩ処理し、そしてランダムプライマーを使用して逆転写した。得られたｃＤＮＡを、転写されないゲノムマウスＤＮＡに特異的なプライマーを使用して、ゲノムの混入の非存在について調べた。ｃＤＮＡを、ＨＰＲＴプライマーを使用して濃度について平均化した。ＲＮＡ転写物は、大脳、皮質、皮質下領域、小脳、脳幹、眼、心臓、肺、肝臓、膵臓、腎臓、皮膚、胆嚢、膀胱、下垂体、副腎、唾液腺、舌、胃、大腸、盲腸、精巣、精巣上体、精嚢、凝固腺、前立腺、卵巣、および子宮において検出可能であった。
【０２１３】
（挙動）
挙動研究について、ホモ接合マウスを以下の通りに作製した：
上記の標的化構築物を、１２９／ＳｖＥｖマウス亜系由来のＥＳ細胞に導入して、キメラマウスを作製した。Ｆ１Ｎ０マウスを、Ｃ５７ＢＬ／６の雌と交配することによって作製した。Ｆ２Ｎ０ホモ接合変異マウスを、Ｆ１ヘテロ接合体の雄と雌を異種交配することによって作製した。Ｆ１Ｎ０ヘテロ接合体を、Ｃ５７ＢＬ／６マウスに戻し交配して、Ｆ１Ｎ１ヘテロ接合体を作製した。Ｆ２Ｎ１ホモ接合マウスを、Ｆ１Ｎ１ヘテロ接合体の雄と雌を異種交配することによって作製した。
【０２１４】
ホモ接合マウスは、以下の挙動表現型を示した：
Ｆ２Ｎ０ホモ接合変異マウスは、ホットプレート試験に対するそれらの応答潜伏時間において、野生型動物と比較して、統計的に有意な増加を示した。ホモ接合マウスは、Ｆ２Ｎ０野生型マウスと比較して、５５℃のホットプレート上に配置される場合に、その後足をなめるまでの時間量において、統計学的に有意な増加を示した。しかし、Ｎ１世代の動物は、いずれの差異も示さなかった。この試験を使用して、侵害障害（すなわち、減少した疼痛応答）を示した。
【０２１５】
Ｎ０およびＮ１世代の両方由来のホモ接合変異動物は、オープンフィールド試験チャンバーの中央領域においてより多くの時間を過ごす傾向を示した。この試験は、不安障害を示し得る。
【０２１６】
（実施例４：ＲＯＲγ遺伝子破壊を含むマウスの作製および分析）
ＲＯＲγの役割を調査するために、ＲＯＲγ遺伝子中の破壊を、相同組換えにより作製した。詳細には、ＲＯＲγ遺伝子中に破壊を含むトランスジェニックマウスを作製した。より詳細には、図９Ａ〜図９Ｂに示されるように、ＲＯＲγ遺伝子（詳細には、配列番号１３を含む）を破壊または改変する能力を有するＲＯＲγ特異的標的化構築物を、本明細書中で配列番号１５または配列番号１６と同定されるオリゴヌクレオチド配列を、この構築物において標的化アーム（相同配列）として使用して作製した。
【０２１７】
この標的化構築物を、１２９／Ｓｖ−＋Ｐ＋Ｍｇｆ−ＳＬＪ／Ｊマウス亜系に由来するＥＳ細胞に導入し、キメラマウスを作製した。Ｆ１マウスを、Ｃ５７ＢＬ／６の雌と交配することによって作製し、そしてＦ２ホモ接合変異マウスを、Ｆ１ヘテロ接合体の雄と雌を異種交配することによって作製した。
【０２１８】
ＲＯＲγ遺伝子中に破壊を含むトランスジェニックマウスを、表現型の変化および発現パターンについて分析した。核レセプター遺伝子中の破壊に関連する表現型を決定した。ホモ接合マウスは、以下の表現型の少なくとも１つを示した：
リンパ節。リンパ節は、全ての雌のホモ接合マウスにおいて存在しなかった。さらに、消化管関連リンパ系組織（ＧＡＬＴ）は、全てのホモ接合変異体由来の腸の慣用的な切片において存在しなかった。
【０２１９】
リンパ球。野生型マウスと比較して、最小から中程度のリンパ球の蓄積が、変異マウスにおける種々の組織（肝臓、肺、膵臓、唾液腺および甲状腺）において存在した。ホモ接合変異マウスは、リンパ腫と一致したリンパ様浸潤を有する多数の器官を有した。この浸潤は、中程度から重篤まで変化し、そして任意の器官系を含み得る。これらの細胞は、増加した有糸分裂速度および頻繁なアポトーシスを有した。併発は、脾臓および胸腺において最も重篤である傾向がある。関与する他の器官としては、以下が挙げられる：肝臓、胆嚢、肺、腎臓、膀胱、心臓、大動脈、骨、骨髄、下垂体、副腎、胸腺、脳および脊髄（髄膜）、膵臓、胃、小腸および大腸、喉頭、気管、食道、舌、骨格筋、精巣、精巣上体、膀胱、精嚢、眼およびハルダー腺。
【０２２０】
２匹の臨床的に病気のホモ接合体の雌（２０７７４および２０２９９）ならびに２匹の臨床的に病気のホモ接合体の雄（１９９２２および２０２９４）は、リンパ球が優勢である非常に上昇した総白血球数を有した。１つの白血病の雌２０７７４において、リンパ球は、未成熟であった（芽細胞性）。これは、リンパ腫の白血病期と一致する。罹患したマウスホモ接合体は、乏しい毛づくろいおよび機能低下を含む病気の徴候を示した。このマウスは、増大した胸部および腹部の寸法、肝脾腫大、拡大した胸腺、および腹水を有した；これらは、弱く、斜視のようであり、背が丸く、悪液性、嗜眠性であり、そして呼吸に障害があった。
【０２２１】
脾臓。以下の表２および図１０において示されるように、全てのホモ接合変異体において、野生型マウスと比較して増加した体重および増加した脾臓：体重の比を含む脾臓における異常が検出された。これらは、平均して、ホモ接合体の雌の０．６１パーセントに対して野生型コントロールの雌の０．４パーセント、およびホモ接合変異体の雄の０．５２パーセントに対して野生型コントロールの雄の０．２６パーセントであった。６８〜１８９日における脾臓重量および脾臓：体重比は、野生型マウスにおけるよりも約５倍大きかった。
【０２２２】
脾臓リンパ様小胞は、胚中心の増加した数のリンパ球（過形成）によって拡大され、そしておよびホモ接合マウス中の小胞の縁領域で拡大された。
【０２２３】
【表２】

肝臓。変異体マウスにおいて、野生型マウスと比較して増加したサイズ、体重、および変色を含む肝臓における異常が検出された。肝臓重量および肝臓：体重比は、野生型コントロールマウスにおけるよりも約２倍大きかった（表２および図１１）。
【０２２４】
腎臓。変異体マウスにおいて、野生型マウスと比較して増加したサイズ、体重、および変色を含む腎臓における異常が検出された。腎臓：体重比は、野生型コントロールマウスにおけるよりも約１．５倍大きかった（表２および図１２）。
【０２２５】
胸腺。胸腺における異常が検出された。胸腺重量および胸腺：体重比は、野生型コントロールマウスにおけるよりも約１０〜３０倍大きかった（表１および図１３）。皮質内側の特徴の減少を有する中程度の胸腺萎縮症が存在した。さらに、胸腺皮質拡大（過形成）および関連した延髄減少（萎縮症）が、全てのホモ接合マウスにおいて観察された。胸腺皮質リンパ球は、増加した有糸分裂活性およびアポトーシスを有する過形成性であった。
【０２２６】
骨および骨髄。骨および骨髄における異常が、６８〜１８９日において検出された。骨髄は、蒼白であり、そして骨は、白い塊が付着し脆性であった。
【０２２７】
体重。雄のホモ接合マウスの体重は、野生型コントロールマウスの体重の約６０％であった。野生型コントロールマウス対雌のホモ接合変異マウスの体重における差異は、存在しなかった。全体の観察は、いくつかのマウスについて不完全であった。
【０２２８】
血清化学。臨床的に病気のホモ接合マウスは、野生型マウスと比較した場合、種々の分析物の上昇を有した。以下を含む複数の肝臓関連分析物を評価した：全４匹の雄および３匹の雌のうちの２匹におけるアラニンアミノトランスフェラーゼ（ＡＬＰ）；２匹の雄におけるビリルビン；ならびに１匹の雄におけるアルカリホスファターゼ（ＡＬＰ）。アスパラギン酸アミノトランスフェラーゼ、別の肝臓関連分析物は、４匹の雄および２匹の雌において上昇した。これらの種々の上昇は、肝臓の腫瘍細胞浸潤と一致している。心臓の腫瘍細胞浸潤もまた、ＡＬＴ上昇に寄与し得た。腎臓関連分析物もまた、上昇した。３匹の雄が、上昇した血液尿素窒素（ＢＵＮ）およびリン酸レベルを有した。これは、おそらく、腎臓および／または腎盤の腫瘍細胞浸潤に関係している。
【０２２９】
発現。総ＲＮＡを、成体Ｃ５７Ｂ１／６野生型マウス由来の器官または組織から単離した。ＲＮＡをＤＮａｓｅＩ処理し、そしてランダムプライマーを使用して逆転写した。得られたｃＤＮＡを、転写されないゲノムマウスＤＮＡに特異的なプライマーを使用して、ゲノムの混入の非存在について調べた。ｃＤＮＡを、ＨＰＲＴプライマーを使用して濃度について平均化した。ＲＮＡ転写物は、小脳、嗅球、眼、心臓、肺、肝臓、膵臓、腎臓、胸腺、リンパ節、皮膚、胆嚢、膀胱、下垂体、副腎、唾液腺、骨格筋、舌、胃、小腸、大腸、盲腸、精巣、精巣上体、精嚢、凝固腺、前立腺、卵巣、および子宮において検出可能であった。
【０２３０】
（実施例５：ＢＭＰ遺伝子破壊を含むマウスの作製および分析）
ＢＭＰの役割を調査するために、ＢＭＰ遺伝子中の破壊を、相同組換えにより作製した。詳細には、ＢＭＰ遺伝子中に破壊を含むトランスジェニックマウスを作製した。より詳細には、図１５Ａ〜図１５Ｂに示されるように、ＢＭＰ遺伝子（詳細には、配列番号１７を含む）を破壊または改変する能力を有するＢＭＰ特異的標的化構築物を、本明細書中で配列番号１９または配列番号２０と同定されるオリゴヌクレオチド配列を、この構築物において標的化アーム（相同配列）として使用して作製した。
【０２３１】
この標的化構築物を、１２９／Ｓｖ−＋Ｐ＋Ｍｇｆ−ＳＬＪ／Ｊマウス亜系に由来するＥＳ細胞に導入し、キメラマウスを作製した。Ｆ１マウスを、Ｃ５７ＢＬ／６の雌と交配することによって作製し、そしてＦ２ホモ接合変異マウスを、Ｆ１ヘテロ接合体の雄と雌を異種交配することによって作製した。
【０２３２】
ＢＭＰ遺伝子中に破壊を含むトランスジェニックマウスを、表現型の変化について分析した。核レセプター遺伝子中の破壊に関連する表現型を決定した。ホモ接合マウスは、以下の表現型の少なくとも１つを示した：
１）異常な尾：
４９日目に、年齢および性が一致した野生型コントロールマウスと比較した場合、調査した９匹のホモ接合マウスのうち５匹（２匹の雌（１０７９６、１０７９９）および３匹の雄（１０８２９、１９４７８、１９４７９））が異常な尾を有した。
【０２３３】
３００日目（実際３０３〜４５０日齢）に、年齢および性が一致した野生型コントロールマウスと比較した場合、９匹のホモ接合変異マウスのうち１匹の雄（１０８２７）が異常な尾を有した。
【０２３４】
２）体重：
４９、９０、１８０、および３００日齢で、低い体重の傾向のホモ接合変異体の雄および雌。
【０２３５】
４９、９０、１８０、および３００日齢で、低い体重／体長比の傾向のホモ接合変異体の雄および雌。
【０２３６】
３）短い体長：
４９、９０、１８０、および３００日齢のホモ接合変異体の雄および雌。
【０２３７】
より詳細には、年齢（４９、９０、１８０、および３００日齢）および性の一致した野生型コントロールマウスと比較した場合、ホモ接合変異マウスは、全ての時点において、３つ全てのパラメーター（体重、体長、および体重／体長比）について低い値を有した。一般的に、ホモ接合変異体と一致した野生型コントロールマウスとの間の差異は、雌についてよりも雄について大きかった。体重および体重／体長比は、１８０日齢後のホモ接合変異体の雄について減少した。各々の３つのパラメーターについて、野生型コントロールの雄とホモ接合変異体の雄との間の差異の大きさは、３００日齢で最大であった。
【０２３８】
（実施例６：気道トリプシン様プロテアーゼ遺伝子破壊を含むマウスの作製および分析）
気道トリプシン様プロテアーゼの役割を調査するために、気道トリプシン様プロテアーゼ遺伝子中の破壊を、相同組換えにより作製した。詳細には、気道トリプシン様プロテアーゼ遺伝子中に破壊を含むトランスジェニックマウスを作製した。より詳細には、図１７Ａ〜図１７Ｂに示されるように、気道トリプシン様プロテアーゼ遺伝子（詳細には、配列番号２１を含む）を破壊または改変する能力を有する気道トリプシン様プロテアーゼ特異的標的化構築物を、本明細書中で配列番号２２または配列番号２４と同定されるオリゴヌクレオチド配列を、この構築物において標的化アーム（相同配列）として使用して作製した。
【０２３９】
この標的化構築物を、１２９／Ｓｖ−＋Ｐ＋Ｍｇｆ−ＳＬＪ／Ｊマウス亜系に由来するＥＳ細胞に導入し、キメラマウスを作製した。Ｆ１マウスを、Ｃ５７ＢＬ／６の雌と交配することによって作製し、そしてＦ２ホモ接合変異マウスを、Ｆ１ヘテロ接合体の雄と雌を異種交配することによって作製した。気道トリプシン様プロテアーゼ遺伝子中に破壊を含むトランスジェニックマウスを、表現型の変化について分析した。
【０２４０】
（実施例７：アクアポリン−８遺伝子破壊を含むマウスの作製および分析）
アクアポリン−８遺伝子の役割を調査するために、アクアポリン−８遺伝子中の破壊を、相同組換えにより作製した。詳細には、アクアポリン−８遺伝子中に破壊を含むトランスジェニックマウスを作製した。より詳細には、図１９Ａ〜図１９Ｂに示されるように、アクアポリン−８遺伝子（詳細には、配列番号２５を含む）を破壊または改変する能力を有するアクアポリン−８特異的標的化構築物を、本明細書中で配列番号２７または配列番号２８と同定されるオリゴヌクレオチド配列を、この構築物において標的化アーム（相同配列）として使用して作製した。
【０２４１】
この標的化構築物を、１２９／Ｓｖ−＋Ｐ＋Ｍｇｆ−ＳＬＪ／Ｊマウス亜系に由来するＥＳ細胞に導入し、キメラマウスを作製した。Ｆ１マウスを、Ｃ５７ＢＬ／６の雌と交配することによって作製し、そしてＦ２ホモ接合変異マウスを、Ｆ１ヘテロ接合体の雄と雌を異種交配することによって作製した。
【０２４２】
アクアポリン−８遺伝子中に破壊を含むトランスジェニックマウスを、表現型の変化および発現パターンについて分析した。核レセプター遺伝子中の破壊に関連する表現型を決定した。ホモ接合マウスは、以下の表現型の少なくとも１つを示した：
体重。ホモ接合変異体の雄は、９０日齢後の野生型コントロールマウスよりもより体重が重い傾向を有する。
【０２４３】
発現。総ＲＮＡを、成体Ｃ５７Ｂ１／６野生型マウス由来の器官または組織から単離した。ＲＮＡをＤＮａｓｅＩ処理し、そしてランダムプライマーを使用して逆転写した。得られたｃＤＮＡを、転写されないゲノムマウスＤＮＡに特異的なプライマーを使用して、ゲノムの混入の非存在について調べた。ｃＤＮＡを、ＨＰＲＴプライマーを使用して濃度について平均化した。ＲＮＡ転写物は、眼、肝臓、膵臓、胸腺、胆嚢、胃、大腸、盲腸、精巣、精嚢、卵巣、および子宮において検出可能であった。
【０２４４】
当業者に明らかなように、上の実施形態の種々の改変が、本発明の精神および範囲から逸脱することなく行なわれ得る。これらの改変およびバリエーションは、本発明の範囲内である。
【図面の簡単な説明】
【図１】
図１は、オーファンアナフィラトキシンＣ３ａレセプターのポリヌクレオチド配列（配列番号１）を示す。図１はまた、アナフィラトキシンＣ３ａレセプターのアミノ酸配列（配列番号２）を示す。
【図２Ａ】
図２Ａは、アナフィラトキシンＣ３ａレセプター遺伝子を破壊するために用いられる標的化構築物の設計を示す。
【図２Ｂ】
図２Ｂは、アナフィラトキシンＣ３ａレセプター遺伝子を破壊するために用いられる標的化構築物の設計を示す。図２Ｂはまた、アナフィラトキシンＣ３ａレセプター標的化構築物において標的化アーム（相同配列）として用いられた、配列番号３および配列番号４として同定される配列を示す。
【図３】
図３は、野生型マウスならびにアナフィラトキシンＣ３ａレセプター遺伝子における破壊を有するヘテロ接合マウスおよびホモ接合マウスの胸腺重量および体重の比に関するグラフを示す。
【図４】
図４は、５−ＨＴ５Ａ遺伝子のポリヌクレオチド配列（配列番号５）を示す。図４はまた、５−ＨＴ５Ａ遺伝子についてのアミノ酸配列（配列番号６）を示す。
【図５Ａ】
図５Ａは、５−ＨＴ５Ａ遺伝子を破壊するために用いられる標的化構築物の設計を示す。
【図５Ｂ】
図５Ｂは、５−ＨＴ５Ａ遺伝子を破壊するために用いられる標的化構築物の設計を示す。図５Ｂはまた、５−ＨＴ５Ａ標的化構築物において標的化アーム（相同配列）として用いられた、配列番号７および配列番号８として同定される配列を示す。
【図６】
図６は、オーファンコルディンのポリヌクレオチド配列（配列番号９）を示す。図６はまた、コルディンポリペプチドのアミノ酸配列（配列番号１０）を示す。
【図７Ａ】
図７Ａは、コルディン遺伝子を破壊するために用いられる標的化構築物の設計を示す。
【図７Ｂ】
図７Ｂは、コルディン遺伝子を破壊するために用いられる標的化構築物の設計を示す。図７Ｂはまた、コルディン標的化構築物において標的化アーム（相同配列）として用いられた、配列番号１１および配列番号１２として同定される配列を示す。
【図８】
図８は、オーファンＲＯＲγのポリヌクレオチド配列（配列番号１３）を示す。図８はまた、ＲＯＲγのアミノ酸配列（配列番号１４）を示す。
【図９Ａ】
図９Ａは、ＲＯＲγ遺伝子を破壊するために用いられる標的化構築物の設計を示す。
【図９Ｂ】
図９Ｂは、ＲＯＲγ遺伝子を破壊するために用いられる標的化構築物の設計を示す。図９Ｂはまた、ＲＯＲγ標的化構築物において標的化アーム（相同配列）として用いられた、配列番号１５および配列番号１６として同定される配列を示す。
【図１０】
図１０は、野生型マウスならびにＲＯＲγ遺伝子における破壊を有するヘテロ接合マウスおよびホモ接合マウスの脾臓重量および体重の比に関するグラフを示す。
【図１１】
図１１は、野生型マウスならびに破壊を有するヘテロ接合マウスおよびホモ接合マウスの肝臓重量および体重の比に関するグラフを示す。
【図１２】
図１２は、野生型マウスならびにＲＯＲγ遺伝子における破壊を有するヘテロ接合マウスおよびホモ接合マウスの腎臓重量および体重の比に関するグラフを示す。
【図１３】
図１３は、野生型マウスならびにＲＯＲγ遺伝子における破壊を有するヘテロ接合マウスおよびホモ接合マウスの胸腺重量および体重の比に関するグラフを示す。
【図１４】
図１４は、ＢＭＰ遺伝子のポリヌクレオチド配列（配列番号１７）を示す。図１４はまた、ＢＭＰポリペプチドのアミノ酸配列（配列番号１８）を示す。
【図１５Ａ】
図１５Ａは、ＢＭＰ遺伝子を破壊するために用いられる標的化構築物の設計を示す。
【図１５Ｂ】
図１５Ｂは、ＢＭＰ遺伝子を破壊するために用いられる標的化構築物の設計を示す。図１５Ｂはまた、ＢＭＰ標的化構築物において標的化アーム（相同配列）として用いられた、配列番号１９および配列番号２０として同定される配列を示す。
【図１６】
図１６は、オーファン気道トリプシン様プロテアーゼのポリヌクレオチド配列（配列番号２１）を示す。図１６はまた、この気道トリプシン様プロテアーゼのアミノ酸配列（配列番号２２）を示す。
【図１７Ａ】
図１７Ａは、気道トリプシン様プロテアーゼ遺伝子を破壊するために用いられる標的化構築物の設計を示す。
【図１７Ｂ】
図１７Ｂは、気道トリプシン様プロテアーゼ遺伝子を破壊するために用いられる標的化構築物の設計を示す。図１７Ｂはまた、気道トリプシン様プロテアーゼ標的化構築物において標的化アーム（相同配列）として用いられた、配列番号２３および配列番号２４として同定される配列を示す。
【図１８】
図１８は、アクアポリン８のポリヌクレオチド配列（配列番号２５）を示す。図１８はまた、アクアポリン８のアミノ酸配列（配列番号２６）を示す。
【図１９Ａ】
図１９Ａは、アクアポリン８遺伝子を破壊するために用いられる標的化構築物の設計を示す。
【図１９Ｂ】
図１９Ｂは、アクアポリン８遺伝子を破壊するために用いられる標的化構築物の設計を示す。図１９Ｂはまた、アクアポリン８標的化構築物において標的化アーム（相同配列）として用いられた、配列番号２７および配列番号２８として同定される配列を示す。[0001]
(Field of the Invention)
The present invention relates to transgenic animals, compositions, and methods related to characterizing gene function.
[0002]
(Background of the Invention)
Laboratory animal models are an important tool for understanding the role of genes. More specifically, the ability to develop animals with specific genes that have been altered or inactivated is invaluable for the study of gene function and is responsible for disease-causing genes with similar expression in humans. And / or unexpected discovery of the mechanism. These genetically engineered animals are also useful for identifying and testing therapeutic treatments for various diseases and disorders.
[0003]
Identifying the functions of many genes is useful in identifying the role of these genes in disease. Due to the high level of homology between humans and mice, it is possible to define the function of individual human genes, for example, by making targeted germline mutations of selected genes in animals. The resulting mutant animal phenotype can be used to help define the phenotype in humans.
[0004]
Several interesting genes have recently been discovered that belong to various families encoding G protein-coupled receptors (GPCRs), chordins, nuclear hormone receptors, bone morphogenetic proteins and aquaporin proteins. Identifying the role of these genes and their expression products may allow the determination of disease pathways and the identification of diagnostically and therapeutically useful targets.
[0005]
GPCRs have been characterized as having seven putative transmembrane domains (designated TM1, TM2, TM3, TM4, TM5, TM6 and TM7). These are thought to represent transmembrane α-helices connected by extracellular or cytoplasmic loops. Most G-protein coupled receptors have one conserved cysteine residue in each of the first two extracellular loops that form disulfide bonds, which are believed to stabilize functional protein structure. G-protein coupled receptors can be bound intracellularly to various intracellular enzymes, ion channels and transporters by heterotrimeric G-proteins. Different G-protein α-subunits preferentially stimulate specific effectors to regulate various biological functions within cells.
[0006]
Anaphylatoxins, especially C3a and C5a, have a variety of biological activities that suggest a role as mediators of the inflammatory response: they include smooth muscle contraction, histamine release, increased capillary permeability, vascular endothelium Causes adherence of leukocytes to leukocytes, chemotaxis of leukocytes and aggregation of platelets and leukocytes (see Vogt, Complement 3: 177-88 (1986)). Most of these effects are supported by the cooperation of other mediators, especially arachidonic acid derivatives, that can be produced by anaphylatoxin-stimulated cells (eg, leukocytes and endothelium). The in vivo effects of complement peptides are largely dependent on their site of production: intravascular release in the systemic circulation, adult respiratory distress, mainly due to activation of leukocytes, aggregation and their accumulation in pulmonary vessels. Causes harmful symptoms such as syndrome and shock lung. Intravascular release can be induced by certain drugs and by blood contact with the surface of the bypass or dialyzer. Activation of the complement system has been reported in various inflammatory disorders and the neurodegenerative processes of the CNS. Recent evidence indicates that complement proteins and receptors are synthesized on or by glial cells and, surprisingly, neurons. Among these proteins, the most likely mediators of the inflammatory function of complement are the receptors for C3a and C5a, chemotactic and anaphylactic peptides. The function of glial cell C3a and C5a receptors (C3aR and C5aR) appears to be similar to C3aR and C5aR in immune cells. However, little is known about the role these receptors have on neurons (see Nataf et al., Trends. Neurosci. 22: 397-402 (1999)).
[0007]
Recently, a cDNA (clone AZ3B) was isolated from a cDNA library of differentiated HL-60 cells using a probe derived from the N-formyl peptide receptor gene (Roglic et al., Biochem. Biophys. Acta. 1305 ( 1-2): 39-43 (1996); accession number U28488; GI: 1977577). The 1.97 kb cDNA encoded a putative G protein-coupled receptor with 482 amino acids. In addition to the putative seven transmembrane domains common to all GPCRs, the protein has a large extracellular loop of approximately 172 amino acids between the fourth and fifth transmembrane domains, (A unique feature of the hundreds of GPCRs identified to date). High sequence homology indicates that this protein and many chemoattractant receptors (eg, the human C3a anaphylatoxin receptor (C3AR) at 170 residues at the amino terminus and 150 residues at the carboxyl terminus (Crass et al., Eur. J. Immunol. 26 (8): 1944-50 (1996)) and the mouse complement C3AR gene (Accession number AF053775; GI: 3170513). Northern and flow cytometric analysis suggested that the message and protein were widely expressed in lung, placenta, heart and endothelial cells in some differentiated hematopoietic cell lines. Have proposed that this protein defines a distinct group of receptors within the GPCR superfamily. Reported a 3.3 kb full-length C3a cDNA encoding an open reading frame of 477 amino acids (J. Immunol. 158: 5277-82 (1997); accession number U77461; GI: 2130534). The putative amino acids contained four putative N-linked glycosylation sites and were 65% identical to the 482 amino acids containing the coding region of the human C3a receptor.
[0008]
One important subfamily of GPCRs is the serotonin neurotransmitter receptor GPCR. Serotonin (aka 5-HT) is an important neuromodulator and a local hormone of the CNS and intestines, mediating a wide range of physiological functions and pathophysiological pathways by activating multiple receptors. Approximately 90% of serotonin in the body is present in chromaffin cells in the intestinal wall, with a small amount present in the myometrial plexus. Serotonin is also found in blood where platelets are present in high concentrations. In the CNS, serotonin neurons originate predominantly in the raphe nuclei of the brainstem and protrude into most regions of the brain. Serotonin is synthesized from the amino acid tryptophan via 5-hydroxytryptophan. In the periphery, 5-HT contacts many smooth muscles, including large blood vessels, the intestine and the uterus, and also induces endothelium-dependent vasodilation via NO formation. Serotonin stimulates sensory nerve endings and is a mediator of peristalsis, and may be involved in platelet aggregation and hemostasis. It may also have implications for inflammatory mediators and microvascular control. In the CNS, serotonin is thought to be involved in a wide range of functions, including control of appetite, mood, anxiety, hallucinations, sleep, vomiting and pain perception. Serotonin receptor ligands find clinical use in the treatment of depression, migraine and post-operative vomiting, and there is strong potential for their use in other conditions.
[0009]
Molecular and pharmacological studies have revealed the existence of multiple subtypes of the 5-HT receptor. One serotonin receptor is the 5-HT-5 receptor. In the CNS, this 5-HT-5 receptor mRNA is found in the cerebellar cortex, hippocampus, zonules, olfactory bulb and cerebellar granular layer. A cDNA (1686 bp) encoding the 5-HT5 (A subtype) receptor was isolated from a mouse brain library (EMBO J. 11 (13), 4779-86 (1992)). This sequence has been submitted to GenBank (GI or NID number: 49758; accession number: Z18278). Comparison of the amino acid sequences revealed that the 5-HT5A receptor is a distant analog of all previously identified 5-HT receptors. Its pharmacological profile is similar to the pharmacological profile of the 5-HT-1D receptor, 5-HT-1D has been shown to inhibit adenylyl cyclase and is predominantly expressed in the basal ganglia , A subtype of the 5-HT receptor. However, unlike the 5-HT-1D receptor, the 5-HT5A receptor did not inhibit adenylation cyclase, and its mRNA was not found in basal ganglia. The coding sequence for the 5-HT5A gene is believed to include bases 509-1582.
[0010]
Over the past 15 years, nearly 350 therapeutic agents targeting seven transmembrane receptors have been successfully introduced to the market. These receptors have an established, proven history as therapeutic targets, but are apparent for the identification and characterization of GPCRs that may play a role in preventing, ameliorating or correcting dysfunction or disease. There is a need.
[0011]
In general, growth factors are proteins that bind to receptors on the cell surface and have the primary consequence of activating cell growth and / or differentiation. Many growth factors are very versatile and stimulate cell division in many different cell types; while others are specific to particular cell types. More specifically, growth factors are substances, such as polypeptide hormones, that affect the growth of a defined animal cell population in vivo or in vitro, but are not nutritional substances. Proteins involved in tissue growth and differentiation can promote or inhibit proliferation and promote or inhibit differentiation, and thus the general term "growth factor" includes cytokines, trophic factors and their inhibitors . Among the growth factors or trophic factors currently known are the transforming growth factors (TGF-α, TGF-β, TGF-γ). Transforming growth factor-β appears to elicit a variety of responses in many different cell types.
[0012]
Among the members of TGF-β are bone morphogenetic proteins (BMPs). BMPs are useful in wound healing, tissue repair, and have been shown to induce cartilage and / or bone growth. BMP has strong effects during embryonic development. One particular member, BMP-4, has been shown to have a strong ventralizing effect in Xenopus embryos, causing blood and mesenchymal differentiation and dorsal aspects such as notochord, muscle and nervous systems. Inhibits tissue formation. (See, for example, Jones et al., Development 115: 639-647 (1991)). BMP-4 is expressed ventrally in Xenopus embryos. BMP-4 expression is increased by ventralization treatment such as irradiation with ultraviolet light. Inhibitors of ventralized BMP are thought to have a dorsalizing effect on tissue differentiation. One such growth factor inhibitor is cordin.
[0013]
Cordin (CHRD) is an important developmental growth factor inhibitor that binds ventralized TGF-β-like BMPs and dorsotypes early vertebrate embryonic tissues by suppressing them to a latent complex. is there. Specifically, CHDR is a secreted BMP antagonist. A sequence database of mammalian cordin sequences was searched to identify ESTs encoding the C-terminal regions of human and mouse CHRD. The full-length cDNA sequence of mouse CHRD was determined to predict a 948 amino acid protein containing four internal cysteine-rich repeats and four potential N-glycosylation sites. (See, eg, Pappano et al., (Genomics 52: 236-239 (1998).) Northern blot analysis detected high levels of the 3.7 kb CHRD transcript in mouse embryos 7 days after mating. The levels of CHDR transcripts are reduced in late developmental and adult tissues, indicating a major role for cordin during gastrulation in mammalian embryos. Both have been shown to be expressed at readily detectable levels in some fetal and adult tissues, especially the liver and cerebellum, suggesting an additional role in tissue formation and homeostasis. Dot blot analysis of mRNA expression in tissue and human fetal tissue showed that CHRD was The partial human CHRD cDNA sequence has been deposited with GenBank (GI or NID: 3822217; accession number AF076612).
[0014]
A human CHRD cDNA encoding a 954 amino acid protein with 86% sequence identity to the mouse protein was cloned and sequenced. (Smith et al., Hum. Genet. 105: 104-111 (1999)). The CHRD gene was also determined to have 23 exons over 11.5 kb. The human CHRD gene was localized to 3q27 by radiation hybrid mapping, and the mouse CHRD gene was localized near chromosome 16 using interspecies backcross. (See Pappano et al., Supra). The genetic organization of the human cordin gene has been determined and is within the 3q27 gene cluster, including THPO (thrombopoietin), CLCN2 (voltage-gated chloride channel gene) and EIF4G1 (eukaryotic translation initiation factor gamma gene). It was shown to be mapped to. (See Smith et al., Supra). The CHRD and THPO genes are very closely adjacent (within a single cosmid clone). These two genes occupy a total of 22 kb and are transcribed from opposite strands using a promoter less than 2 kb apart. The CHRD gene and the cordin-regulated GSC (goosecoid) gene were found to be potential candidate genes for the developmental malformation syndrome Cornelia de Lange syndrome (CDLS), which maps to 3q. The syndrome is mainly characterized by mental handicaps, growth retardation, unique facial features and limb-reduction defects.CDLS patients typically occur as sporadic cases, Some reports suggest dominant inheritance.CHRD and GSC gene candidates are supported by several lines of evidence, including: the CDLS gene at 3q26.3-q27. Previous evidence for a significant link between CDLS and thrombocytopenia Report suggesting a run; Suspicious genetic heterogeneity in CDLS; Location of the GSC gene in close proximity to the 14q32 breakpoint, detected in CDLS patients with a novel equilibrium translocation; Known regulation of cordin expression by Guscoid And the embryonic expression pattern of the mouse GSC gene, but mutation screening failed to identify CDLS patient-specific mutations in CHRD or GSC.
[0015]
Murine CHRD mRNA is first expressed in the anterior primordial striatum and then in the mesendoderm from the nodal and axial stems from which CHRD may play a role in early embryo patterning Suggest that. Targeted inactivation of CHRD resulted in stillborn animals with normal early development and neural induction, but with late defects in inner and outer ear development and abnormalities in pharyngeal and cardiovascular organization.
[0016]
In midgut, it was demonstrated that noggin expression overlaps with CHRD expression. (See, e.g., Bachiller et al., Nature 403: 658-661 (2000).) Noggin mutants undergo normal gastrulation and anterior central nervous system patterning, but in late stages, many Abnormalities were observed in the posterior spinal cord and somites.A cross between mice heterozygous for the noggin and cordin mutations did not generate double homozygous mutants in neonates. Cordin double-null embryos were found in animals dissected shortly after birth, both reabsorbed, but with apparent panforencephaly, single nasal fossa, monocular and jaw disorders. These malformations, not observed in any of the mutations themselves, were found in embryos lacking sonic hedgehog (shh). In 12.5 day embryos, double mutant embryos were harvested with a more severe phenotype similar to aprosencephaly, and dissected at 8.5 day embryos. Forebrain shrinkage was clearly evident in heavy mutant embryos, indicating that cordin and noggin are not required for the establishment of anterior visceral endoderm but for subsequent finishing of the anterior pattern Mesodermal development was also affected, indicated by the lack of shh, suggesting that the BMP antagonists cordin and noggin complement each other during early mouse development. When done, anterior-posterior, dorsal-ventral, and left-right patterning were all affected.
[0017]
Recently, a 3254 bp sequence of the mouse CHRD cDNA was reported (GenBank accession number AF0976276; GI 4406185). This coding region spanned nucleotides 1-2847 and was thought to encode a 948 amino acid polypeptide. In view of the importance of growth factor inhibitors, there is a clear need for the identification and characterization of growth factor inhibitors that can play a role in preventing, ameliorating or correcting dysfunction or disease.
[0018]
The nuclear receptor superfamily comprises a diverse group of ligand-activated transcription factors that share a common modular structure. This superfamily includes a number of receptors, such as steroid hormone, thyroid hormone, retinoid and vitamin D receptors, and olfactory receptors for which no ligand has been identified. This superfamily can be divided into a number of subfamilies, each containing several closely related genes. In addition to RORα and RORβ, a third member of the ROR subfamily, RORγ, has been cloned (Hirose et al., Biochem. Biophys. Res. Commun. 205: 1976-1983 (1994); Medvedev et al., Gene 181: 199-206 (1996); Medvedev et al., Genomics, 46: 93-102 (1997)). The amino acid sequence of RORγ is highly conserved among species; the amino acid sequence between mouse RORγ and human RORγ shows 88% identity (Hirose et al., (1994); Medvedev et al., (1996). Medvedev et al., (1997)).
[0019]
Most nuclear receptors contain five major domains: the amino-terminal A / B domain, a highly conserved DNA binding (C) domain containing two zinc finger domains, a hinge region (D), and a ligand binding domain. (E) and the carboxyl-terminal F domain. The nuclear receptor is monomeric with respect to the consensus sequence PuGGTCA of the core motif separated by one or more nucleotides or a response element composed of a single core motif, including tandem, palindromic or inverted repeats. Binds as a dimer or homodimer or heterodimer. The role of nuclear receptors has been demonstrated in the control of embryonic development, cell proliferation and differentiation. In addition, genetic alterations in the expression and structure of these receptors have been implicated in various disease processes (Hughes et al., Science 242: 1702-1705 (1988); DeLucas, FASEB J. 5: 2924-2933 (1991). ); Chomienne et al., Leukemia 4: 802-807 (1991)).
[0020]
Recently, cDNA (mRORγ orphan nuclear receptor) mRNA has been isolated from a mouse muscle cDNA library (Medvedev et al., Gene 181 199-206 (1996); accession number U4350; GI: 1155340). The 2066 bp cDNA encoded a 516 amino acid residue protein similar to the human 560 amino acid ROR (see Hirose et al., Biochem. Biophys. Res. Commun. 205: 1976-83 (1994)). In addition, there was high sequence homology between this protein and human RORγ with an overall identity of 88%. Analysis of the RORγ response element using RORγ synthesized in vitro shows that it binds as a monomer to a response element composed of a single core motif GGTCA preceded by a 6 bp AT-rich sequence. Revealed. Northern blot analysis using RNA from different tissues showed that mRORγ was found to be highly expressed in skeletal muscle, liver and kidney.
[0021]
Thus, important regulatory functions have been generally suggested for members of the ROR subfamily (Austin et al., Cell Growth & Differentiation, 9: 267-276 (1998)), but the specific role played by RORγ in development and differentiation. Still needs to be elucidated.
[0022]
Among the members of TGF-β are bone morphogenetic proteins (BMPs). BMPs are useful in wound healing, tissue repair, and have been shown to induce cartilage and / or bone growth. BMP has strong effects during embryonic development. One particular member, BMP-4, has been shown to have a strong ventralizing effect in Xenopus embryos, causing blood and mesenchymal differentiation and dorsal aspects such as notochord, muscle and nervous systems. Inhibits tissue formation (see, for example, Jones et al., Development 115: 639-647 (1991)). BMP-4 is expressed ventrally in Xenopus embryos. BMP-4 expression is increased by ventralization treatment such as irradiation with ultraviolet light.
[0023]
During animal development, cells exchange signals with cells next to them, which signals specific groups of cells to organize differentiated structures (eg, limbs, nodes or other parts of the body). ) Is generated. Understanding these processes may allow to regenerate damaged body parts or manipulate cells to diagnose birth defects. Twisted gastrulation protein (TSG) is one of five known secreted proteins required for patterning the dorsal part of early Drosophila embryos. Unlike the decapentaplegic (DPP) protein, which is required for patterning the entire dorsal half of the embryo, TSG is only required for determining the developmental fate of cells in the dorsal midline. When expressed in ventral striped cells, the TSG protein can spread to most dorsal cells and rescue dorsal midline cells in tsg mutant embryos. The function of TSGs in a permissive rather than supportive role, which determines the developmental fate of cells along the dorsal midline after peak levels of DPP activity, "primes" cells to respond to TSGs "
[0024]
Serine proteases are secreted proteins that contain an N-terminal signal peptide that serves to transport the immature protein through the endoplasmic reticulum and is then cleaved (von Heijne, Nutl. Acid. Res. 14: 5683-90). (1986)). Differences in these signal sequences provide one means of identifying individual enzymes. Some serine proteases, especially digestive enzymes, exist as inactive precursors or preproenzymes, and contain a leader or activation peptide sequence 3 'to the signal peptide. Other features that distinguish the various serine proteases include the presence or absence of N-linked glycosylation sites that provide a membrane anchor, the number and distribution of cysteine residues that determine the secondary structure of the serine protease, and S ′. Sequence of the substrate binding site. This S 'substrate binding region is defined by residues ranging from approximately +17 to +29 relative to the N-terminal I (+1). Differences in this region of the molecule are thought to determine the substrate specificity of the enzyme.
[0025]
Proteases are involved in various developmental and metabolic processes (Strood, Sci. Am. 231: 74-88 (1974); Neuroth, Science 224: 350-357 (1984)). Molecular deficiencies that alter enzyme function often result in severe human disease, such as bleeding, thrombosis, and atherosclerosis. Hepsin is a novel serine protease of the trypsin family and contains one transmembrane domain near the amino terminus (Kurachi et al., Methods in Enzymology, 244: 100-114 (1994)). This structural feature distinguishes hepsin from most other serine proteases. In addition to hemagglutination, hepsin is reported to be important for cell proliferation. Transgenic mice with a disrupted hepsin gene show elevated serum alkaline phosphatase levels (US Pat. No. 5,981,830; Wu et al., J. Clin. Invest. 101: 321-326 (1998)). On histological examination, no obvious abnormalities were found in the major organs of the hepasin − / − mice. This result indicated that hepsin was not essential for embryonic development and normal hemostasis.
[0026]
In the Drosophila Drosophila meranogaster, another transmembrane serine protease, Stable-stubbloid, has been reported to have structural features similar to those of hepsin. Stable-stubbleid proteins play an important role in epithelial morphogenesis and development in Drosophila. Defects in the Stable-stubbloid gene result in leg, wing and bristle malformations (Apple et al., Proc. Natl. Acad. Sci. USA 90: 4937-4941 (1993)).
[0027]
Airway trypsin-like protease is an enzyme isolated from the sputum of patients with chronic airway disease (Yamaoka et al., J. Biol. Chem. 273 (19): 11895-901 (1998); accession number NP 004253; GI : 4758508). The complete 1517 base pair cDNA sequence was obtained with an open reading frame encoding a polypeptide of 418 amino acid residues. This polypeptide has a putative hydrophobic transmembrane domain near the HN2 terminus and consists of a catalytic region of 232 residues and a specific catalytic region of 186 residues. This polypeptide is suggested to be a type II transmembrane protein with a COOH-terminal catalytic region extracellular. This protein was synthesized as a membrane-bound precursor and was thought to mature by limited proteolysis into a soluble and active protease. It showed 29-38% identity in the sequence of the catalytic domain to human hepsin, enteropeptidase, acrosin and mast cell tryptase. The non-catalytic region had little similarity to other known proteins. In Northern blot analysis, the 1.9 kilobase transcript was the most prominent detectable in the trachea among the 17 human tissues tested. Recently, a mouse EST with sequence similarity to human airway trypsin-like protease and human hepsin was identified from the Knowles Solter embryo cDNA library (Accession number AA415658; GI: 2075952).
[0028]
The aquaporin family is a group of selective water channels and is part of a larger family of major integral membrane proteins (MIPs). To date, eight different aquaporins have been cloned. Aquaporin-1 through aquaporin-4 have been identified in the kidney and play a role in the water reabsorption of glomerular filtration along nephrons. Aquaporin 0 is the major (60%) endogenous protein of the lens fiber cells of the eye. Mutations in this gene are associated with cataract formation in mice. Aquaporin 1, also called CHIP-28, is present in the membrane as a homotetramer and is present in red blood cells, choroid plexus, proximal tubules and descending limb of the Henle trap in the kidney, and in many other sites (Heymann et al., JSB 121 (2), 191-206 (1998)). Surprisingly, no pathological consequences are known in patients lacking a functional AQP4 gene. Aquaporin 2, also called WCH-CD, is the major cellular water channel of the cortical and medullary collecting ducts and was translocated to the apex of the membrane by synaptobrevin or vesicle-associated membrane protein 2 (VAMP2). In some cases, unless arginine vasopressin acts, it is localized in cytoplasmic vesicles. Lack of a functional AQP2 protein results in the rare formation of nephrogenic diabetes insipidus. AQP3 and AQP4 are located in the basolateral membrane of the major cells of the collecting duct, and AQP3 is predominantly located in the cortex or outside the medulla, while AQP4 is predominant in the inner medulla collecting duct. The clinical significance of these two water channels in kidney water preservation has not yet been determined. Recently, transgenic mice lacking AQP4 have been generated (Ma et al., J. Clin. Invest. 100: 957-962 (1997)). Interestingly, the phenotypic abnormality is very subtle, with small defects in maximal urine concentration, consistent with the predominant localization of AQP4 in the lower part of the IMCD. Aquaporin-5 is clearly associated with fluid secretion in exocrine tissues. Although the exact function of aquaporin-6 is not known due to its uncertain localization, its limited presence in the kidney may suggest a potential role in water transport. Aquaporin-7 appears to play a role in cryopreservation of sperm, while aquaporin-8 is thought to be responsible for pancreatic juice secretion (Dibas et al., Proc. Soc. Exp. Biol. Med. 219: 183-99). (1998). In general, except for the rat, few studies have examined the ontogeny of these proteins, and virtually nothing is known about the expression of these genes in pregnancy or any impairment of fluid balance in the mother or fetus. Not.
[0029]
Recently, a mouse aquaporin-8 water channel cDNA has been isolated (accession number AF018952; GI: 2353796). The 1447 bp cDNA (designated AQP8) was found to have a 783 bp open reading frame. The encoded 261 amino acid hydrophobic protein contains a conserved NPA motif found in MIP family members (Ma et al., Biochem. Biophys. Res. Commun. 240: 324-8 (1997)).
[0030]
(Summary of the Invention)
The present invention relates generally to transgenic animals, as well as compositions and methods for characterizing gene function.
[0031]
The invention provides a transgenic cell that contains a disruption in a target gene. The transgenic cells of the present invention include any cells capable of performing homologous recombination. Preferably, the cells of the invention are stem cells, and more preferably, embryonic stem (ES) cells, and most preferably, mouse ES cells. According to one embodiment, transgenic cells are produced by introducing a targeting construct into a stem cell and generating a homologous recombinant that results in a mutation in the target gene. In another embodiment, the transgenic cells are from a transgenic animal described below. Cells from a transgenic animal include cells isolated from or present in tissues or organs, and any cell line or any progeny thereof.
[0032]
The invention also provides targeting constructs and methods of producing targeted constructs that, when introduced into a stem cell, produce a homologous recombinant. In one embodiment, a targeting construct of the invention comprises first and second polynucleotide sequences that are homologous to a target gene. The targeting construct also includes a polynucleotide sequence encoding a selectable marker that is preferably located between two different homologous polynucleotide sequences in the construct. The targeting construct can also include other regulatory elements that can enhance homologous recombination.
[0033]
The invention further provides non-human transgenic animals and methods of producing such non-human transgenic animals that contain a disruption in the target gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the target gene. In one aspect, the transgenic animal of the present invention lacks the function of the target gene. In another aspect, the transgenic animals of the invention include a phenotype associated with having a mutation in a target gene.
[0034]
The invention also provides a method of identifying an agent that can affect a phenotype associated with disruption of a target gene in a transgenic animal. For example, a putative drug is administered to a transgenic animal, and the transgenic animal's response to the putative drug is measured and compared to the response of a "normal" or wild-type mouse, or a transgenic animal control (no drug treatment). ). The present invention further provides an agent identified according to such a method. The present invention also provides a method of identifying an agent useful as a therapeutic agent for treating a condition associated with disruption of a target gene.
[0035]
The present invention further provides a method for identifying an agent having an effect on the expression or function of a target gene. The method involves administering to the transgenic animal, preferably a mouse, an effective amount of the drug. The method includes, for example, measuring the response of the transgenic animal to the drug, and comparing the response of the transgenic animal to a control animal (which can be, for example, a wild-type animal or a transgenic animal control). . Compounds that may have some effect on target gene expression or function can also be screened against cells in a cell-based assay, for example, to identify such compounds.
[0036]
The invention also provides a cell line comprising the nucleic acid sequence of the target gene. Such cell lines may be capable of expressing such sequences by operable linkage to a promoter that functions in the cell line. Preferably, the expression of the target gene sequence is under the control of an inducible promoter. Also provided is a method of identifying an agent that interacts with a target gene, comprising the steps of contacting a target gene or target protein with an agent and detecting the agent / target gene complex or agent / target protein complex. Such a complex can be detected, for example, by measuring the expression of an operably linked detectable marker.
[0037]
The present invention further provides a method of treating a disease or condition associated with a disruption in a target gene, more particularly a disruption in the expression or function of a target gene or a target protein encoded by the target gene. In a preferred embodiment, the method of the invention comprises administering to a patient in need thereof a therapeutic agent that results in expression or function of the target gene or protein, a disease associated with disruption in the expression or function of the target gene. Or treating the condition. According to this embodiment, the method comprises administering a therapeutically effective amount of a natural, synthetic, semi-synthetic, or recombinant target gene, target gene product or fragment thereof, and a natural, synthetic, semi-synthetic or recombinant analog. including.
[0038]
The invention further provides a method of treating a disease or condition associated with the expression or function of a disrupted target gene, comprising detecting the mutated target gene and replacing it by gene therapy. Is included.
[0039]
(Definition)
The term "gene" refers to (a) a gene comprising at least one of the DNA sequences disclosed herein; (b) any encoding the amino acid sequence encoded by the DNA sequences disclosed herein. And / or (c) any DNA sequence that hybridizes to the complement of the coding sequence disclosed herein. Preferably, the term includes all sequences necessary for normal gene expression, including coding and non-coding regions, and preferably including promoters, enhancers and other regulatory sequences.
[0040]
The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to a polymeric form of nucleotides of any length. A polynucleotide may include deoxyribonucleotides, ribonucleotides and / or analogs thereof. Nucleotides can have any three-dimensional structure and can perform any function, known or unknown. The term "polynucleotide" includes single-stranded, double-stranded and triple-helical molecules.
[0041]
“Oligonucleotide” refers to a single-stranded or double-stranded DNA polynucleotide of 5 to about 100 nucleotides. Oligonucleotides are also known as oligomers or oligos, and can be isolated from a gene or chemically synthesized by methods known in the art. "Primer" refers to an oligonucleotide, usually single-stranded, that provides a 3'-hydroxy terminus for initiation of enzyme-mediated nucleic acid synthesis. The following are non-limiting embodiments of polynucleotides: gene or gene fragment, exon, intron, mRNA, tRNA, rRNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence DNA, RNA of any sequence, nucleic acid probe and primer. Nucleic acid molecules can also include modified nucleic acid molecules such as methylated nucleic acid molecules and nucleic acid molecule analogs. Purine and pyrimidine analogs are known in the art and include aziridinyl cytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- Carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, -Methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluuracil and 2,6-diaminopurine. The use of uracil as an alternative to thymine in deoxyribonucleic acids is also considered an analog form of pyrimidine.
[0042]
A "fragment" of a polynucleotide is a polynucleotide comprising at least 9 contiguous nucleotides of a coding or non-coding sequence, preferably at least 15 contiguous nucleotides, and more preferably at least 45 nucleotides.
[0043]
The term "gene targeting" occurs when a fragment of genomic DNA is introduced into a mammalian cell, and refers to one type of homologous recombination in which the fragment is located at an endogenous homologous sequence and undergoes recombination. .
[0044]
The term "homologous recombination" refers to the exchange of a DNA fragment between two DNA molecules or between chromatids at the site of a homologous nucleotide sequence. As used herein, the term "homology" refers to at least about 70% sequence identity, typically at least about 85% sequence identity, preferably at least about 95%, relative to a reference sequence. %, And more preferably about 98% sequence identity, and most preferably about 100% sequence identity compared to a reference sequence. Homology may be determined using the "BLASTN" algorithm. It is understood that homologous sequences can have insertions, deletions and substitutions in the nucleotide sequence. Thus, the linear sequence of nucleotides can be essentially identical, even if some of the nucleotide residues do not correspond exactly or are not aligned. A reference sequence can be a portion of a gene or flanking sequence, or a subset of a larger sequence, such as a repeated portion of a chromosome.
[0045]
The term "target gene" (or alternatively referred to as "target gene sequence" or "target DNA sequence" or "target sequence") refers to any nucleic acid molecule or homologous gene that is modified by homologous recombination. Refers to nucleotides. The target sequence may be an intact gene, exon or intron, regulatory sequence or any region between the genes. The target gene comprises a particular gene or a portion of a locus in individual genomic DNA. As provided herein, a target gene of the present invention is an anaphylatoxin C3a receptor gene, a 5-HT5A gene, a RORγ gene, a BMP gene, an airway trypsin-like protease gene, or an aquaporin 8 gene.
[0046]
"Anaphylatoxin C3a receptor gene" refers to a sequence comprising SEQ ID NO: 1 or a sequence comprising the sequence identified in Genebank with accession numbers U77461; GI2130534. In one aspect, the coding sequence for the anaphylatoxin C3a receptor gene comprises SEQ ID NO: 1 or the gene identified in Genebank with accession numbers U77461; GI2130534.
[0047]
"5-HT5A gene" refers to a sequence comprising SEQ ID NO: 5 or a sequence comprising the sequence identified in Genebank with accession number Z18278; GINO: 49758. In one aspect, the coding sequence for the 5-HT5A gene comprises SEQ ID NO: 5 or the gene identified in Genebank with accession number Z18278; GINO: 49758.
[0048]
"Cordin gene" refers to a sequence comprising SEQ ID NO: 9 or a sequence comprising the sequence identified in Genebank with accession numbers AF0976276; GI4406185. In one aspect, the coding sequence of the cordin gene comprises SEQ ID NO: 9 or the gene identified in Genebank with accession numbers AF096276; GI4406185.
[0049]
"RORγ gene" refers to a sequence comprising SEQ ID NO: 13 or a sequence comprising the sequence identified in Genebank with accession number U4350; GINO: 1155340. In one aspect, the coding sequence of the RORγ gene comprises SEQ ID NO: 13 or the gene identified in Genebank with accession number U4350; GINO: 1155340.
[0050]
"BMP gene" refers to a sequence comprising SEQ ID NO: 17 or a sequence comprising the sequence identified in Genebank with accession numbers AF292033; GINO: 9837569. In one aspect, the coding sequence of the BMP gene comprises SEQ ID NO: 17 or the gene identified in Genebank with accession numbers AF292203; GINO: 9837569.
[0051]
“Airway trypsin-like protease gene” refers to a sequence comprising SEQ ID NO: 21 or a sequence comprising the sequence identified in Genebank with accession numbers AA415658; GINO: 2075952. In one aspect, the coding sequence of the airway trypsin-like protease gene comprises the airway trypsin-like protease gene identified in SEQ ID NO: 21 or Genebank with accession numbers AA415658; GINO: 20759923.
[0052]
"Aquaporin 8 gene" refers to a sequence comprising SEQ ID NO: 25 or a sequence comprising the sequence identified in Genebank with accession numbers AF0188952; GINO: 2353796. In one aspect, the coding sequence for the aquaporin 8 gene comprises the aquaporin 8 gene identified in SEQ ID NO: 25 or Genebank with accession numbers AF0188952; GINO: 2353796.
[0053]
"Disruption" of the target gene occurs when a fragment of genomic DNA is located at an endogenous homologous sequence and recombination occurs. Disruptions or modifications of these sequences can include insertions, missenses, frameshifts, deletions, or substitutions or replacements of the DNA sequence, or any combination thereof. Insertions include insertions of entire genes, which can be of animal, plant, prokaryotic, or viral origin. Disruption can, for example, alter or replace the promoter, enhancer, or splice site of the target gene and partially or completely inhibit the production of the normal gene product, or By enhancing the activity of the product, the normal gene product can be altered.
[0054]
The term "transgenic cell" refers to a cell that contains, in its genome, a target gene that has been completely, partially, disrupted, modified, altered, or replaced by a method of gene targeting. Say.
[0055]
The term "transgenic animal" refers to an animal whose genome contains a particular gene that has been disrupted by a method of gene targeting. The transgenic animals include both heterozygous animals (ie, one defective allele and one wild-type allele) and homozygous animals (ie, two defective alleles).
[0056]
As used herein, the term "selectable marker" or "positive selectable marker" refers to a product that, under certain conditions, allows only cells carrying this gene to survive and / or proliferate. Refers to the gene that encodes For example, introduced neomycin resistance (Neomycin) ^r )) Plant and animal cells that express the gene are resistant to compound G418. This Neo ^r Cells that do not carry the genetic marker are killed by G418. Other positive selectable markers are known to those of skill in the art.
[0057]
"Host cell" includes an individual cell or cell culture that may be or has been the recipient for vectors or for uptake of nucleic acid molecules and / or proteins. A host cell contains the progeny of a single host cell, and the progeny will not necessarily be completely (in morphology or in the entire DNA complement) due to natural, accidental, or deliberate mutations in the original parent. Need not be the same. Host cells include cells transfected with a construct of the present invention.
[0058]
The term "modulate," as used herein, refers to inhibiting, reducing, increasing, reducing the function, expression, activity, or phenotype of a target gene or protein associated with disruption in the target gene. Or refers to enhancement.
[0059]
The term "ameliorate" refers to the deceasing, reducing or eliminating of a condition, disease, disorder or phenotype, including abnormalities or symptoms associated with a disruption in a target gene.
[0060]
The term “abnormal” refers to any disease, disorder, condition or phenotype (including pathological conditions) that involves disruption of a target gene.
[0061]
(Detailed description of the invention)
The present invention is based, in part, on the evaluation of the expression and function of genes and gene expression products, primarily those genes and gene expression products associated with a target gene. In particular, the invention allows the definition of disease pathways and the identification of diagnostically and therapeutically useful targets. For example, a gene that is mutated or down-regulated in a disease state may be associated with causing or exacerbating the disease state. Treatments involving up-regulating the activity of such genes, or involving alternative pathways, may ameliorate the disease state.
[0062]
(Generation of targeting construct)
The targeting constructs of the invention can be produced using standard methods known in the art (eg, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring). Harbor, New York; Ed., EN Glover, 1985, DNA Cloning: A Practical Approach, Volumes I and II; Ed., MJ. Gait, 1984, Oligonucleotide Synthesis; BD Hames and S. J. Higgins eds., 1985, Nucleic Acid Hybridization; BD Hames and SJ Higgins. Ed., 1984, Transcription and Translation; RI Freshney, ed., 1986, Animal Cell Culture; Immobilized Cells and Enzymes, IRL Press, 1986; , 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). For example, targeting constructs can be prepared according to conventional methods, wherein the sequences can be synthesized, isolated from natural sources, engineered, cloned And may be subjected to in vitro mutagenesis, primer repair and the like. At various stages, the ligated sequences can be cloned and analyzed by restriction analysis, sequencing, and the like.
[0063]
Targeting DNA can be constructed using techniques well known in the art. For example, targeted DNA can be chemically synthesized oligonucleotides, nicked translation of a double-stranded DNA template, polymerase chain reaction amplification (or ligase chain reaction amplification) of a sequence, a sequence of interest (eg, cloned cDNA or genomic DNA). Prokaryotic or targeted cloning vectors (e.g., plasmids, phagemids, YACs, cosmids, bacteriophage DNA, other viral DNAs or replication intermediates, or synthetic DNA, or any combination of the foregoing). Purification of purified restriction fragments) and other sources of single- and double-stranded polynucleotides having the desired nucleotide sequence. Further, the length of homology can be selected using methods known in the art. For example, the choice may depend on the sequence composition and sequence complexity of the predetermined endogenous target DNA sequence.
[0064]
The targeting construct of the present invention typically comprises a first sequence homologous to a portion or region of a target gene, and a second sequence homologous to a second portion or region of the target gene. The targeting construct further comprises a positive selectable marker, preferably located between the first and second DNA sequences homologous to a portion or region of the target DNA sequence. Is done. The positive selectable marker can be operably linked to a promoter and a polyadenylation signal.
[0065]
Other regulatory sequences known in the art can also be incorporated into the targeting construct and disrupt or control the expression of a particular gene in a particular cell type. Additionally, the targeting construct may also include a sequence encoding a screening marker, such as green fluorescent protein (GFP), or another modified fluorescent protein.
[0066]
The size of the homologous sequence is not critical and can range from as little as 50 base pairs to as much as 100 kb, but preferably each fragment is longer than about 1 kb, more preferably between about 1 kb and about 10 kb. And even more preferably between about 1 kb and about 5 kb. One skilled in the art will recognize that longer fragments may increase the number of homologous recombination events in ES cells, but longer fragments are also more difficult to clone.
[0067]
In a preferred embodiment of the present invention, the targeting construct is identified in pending US patent application Ser. No. 08 / 971,310, filed Nov. 17, 1997, the disclosure of which is hereby incorporated by reference in its entirety. Prepared directly from a plasmid genomic library using the method described in Generally, the sequence of interest is identified and isolated from a plasmid library in one step using, for example, long range PCR. After isolation of this sequence, a second polynucleotide that disrupts the target sequence can be readily inserted between the two regions encoding the sequence of interest. In accordance with this aspect, the construct is generated in two steps: (1) amplifying a sequence homologous to the target sequence (eg, using long range PCR); Inserting the additional polynucleotide (eg, a selectable marker) into the PCR product such that nucleotides are flanked by the homologous sequence. Typically, this vector is a plasmid from a plasmid genomic library. The completed construct is also typically a circular plasmid.
[0068]
In another embodiment, the targeting construct is described in US application Ser. No. 60 / 232,957, filed Sep. 15, 2000, the disclosure of which is incorporated herein in its entirety. It is designed according to the adjusted positive selection method. This targeting construct has two lacO sites and is located under the PGK promoter, a PGK-neo fusion gene, and an NLS-lacI gene containing a lac repressor fused to a sequence encoding NLS from the SV40 T antigen. It is designed to include
[0069]
In another embodiment, the targeting construct may include more than one selectable marker gene, including a negative selectable marker, such as the herpes simplex virus tk (HSV-tk) gene. The negative selectable marker can be operably linked to a promoter and a polyadenylation signal (eg, US Pat. No. 5,464,764; US Pat. No. 5,487,992; US Pat. No. 5,627,059). And U.S. Patent No. 5,631,153).
[0070]
(Confirmation of cell generation and homologous recombination events)
Once a suitable targeting construct has been prepared, the targeting construct can be introduced into a suitable host cell using any method known in the art. A variety of techniques can be used in the present invention, including, for example, pronuclear microinjection; retroviral-mediated gene transfer into the germline; gene targeting in embryonic stem cells; electroporation of the embryo; And calcium phosphate / DNA co-precipitation, microinjection of DNA into the nucleus, bacterial protoplast fusion with intact cells, transfection, polycations (eg, polybrene, polyornithine, etc.) and the like (eg, US Patent Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82: 6148-6152; Thompson et al., 1989, Cell 56: 313-321; Lo, 1983, Mo. 1 Cell.Biol. 3: 1803-1814; See Lavitrano et al., 1989, Cell, 57: 717-723). Various techniques for transforming mammalian cells are known in the art (eg, Gordon, 1989, Intl. Rev. Cytol., 115: 171-229; Keown et al., 1989, Methods in Enzymology; Keown). Et al., 1990, Methods and Enzymology, 185, 527-537; Mansour et al., 1988, Nature, 336: 348-352).
[0071]
In a preferred aspect of the invention, the targeting construct is introduced into a host cell by electroporation. In this process, a high field strength electric shock renders the biological membrane reversibly permeable, allowing the introduction of this construct. The pores created during electroporation allow for uptake of macromolecules (eg, DNA) (eg, Potter, H. et al., 1984, Proc. Nat'l. Acad. Sci. US). A. 81: 7161-7165).
[0072]
Any cell type capable of homologous recombination can be used in the practice of the present invention. Examples of such target cells include vertebrates, including mammals such as humans, bovine species, sheep species, mouse species, monkey species, and filamentous fungi and higher multicellular organisms (eg, plants). Examples include cells derived from other eukaryotic organisms.
[0073]
Preferred cell types include embryonic stem (ES) cells, which are typically obtained from pre-implantation embryos cultured in vitro (eg, Evans, MJ et al., 1981, Nature 292: 154-156; Bradley, MO et al., 1984, Nature 309: 255-258; Gossler et al., 1986, Proc. Natl.Acad.Sci.USA 83: 9065-9069; and Robertson et al., 1986, Nature 322: 445-448). ES cells are cultured and prepared for introduction of the targeting construct using methods well known to those of skill in the art (eg, Robertson, EJ, ed. “Teratocarcinomas and Embronic Stem Cells, a Practical Approach”). , IRL Press, Washington DC, 1987; Bradley et al., 1986, Current Topics in Devel. Biol. 20: 357-371; Hogan et al., "Manipulating the Mouser Carrying Embryo": A.R. Cold Spring Harbor NY, 1986; Thomas et al. 1987, Cell 51: 503; Koller et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 10730; Dorin et al., 1992, Transgenic Res. 1: 101; and Veis et al., 1993, Cell 75: 229. That). ES cells into which the targeting construct is inserted are derived from an embryo or blastocyst of the same species as the developing embry into which the targeting construct is introduced. ES cells are typically selected for their ability to integrate into the inner cell mass and to contribute to the individual's germ line when introduced into a mammal, an embryo at the blastocyst stage of development. Thus, any ES cell line having this capability is suitable for use in practicing the present invention.
[0074]
The present invention can also be used to knock out genes from other cell types (eg, stem cells). By way of example, the stem cells can be bone marrow, lymphatic or neural progenitor cells and precursor cells. These cells containing gene disruptions or knockouts may be particularly useful in studying target gene function in ontogenetic pathways. Stem cells can be derived from any vertebrate species (eg, mouse, rat, dog, cat, pig, rabbit, human, non-human primate, etc.).
[0075]
After the targeting construct has been introduced into the cells, cells in which successful gene targeting has occurred are identified. Insertion of the targeting construct for the targeted gene is typically detected by identifying cells for expression of the marker gene. In a preferred embodiment, cells transformed with the targeting constructs of the present invention are subjected to treatment with a suitable agent that selects for cells that do not express the selectable marker. Only cells that express the selectable marker gene will survive and / or proliferate under certain conditions. For example, cells that express the introduced neomycin resistance gene are resistant to compound G418, but cells that do not express this neo gene marker are killed by G418. If the targeting construct also contains a screening marker (eg, GFP), homologous recombination can be identified through screening of cell colonies under fluorescent light. Cells that have undergone homologous recombination are deficient in the GFP gene and do not fluoresce.
[0076]
When a regulated positive selection method is used in the identification of homologous recombination events, expression of the selectable marker gene is inhibited after random incorporation, but its expression is permissive (dissuppression) after homologous recombination. This targeting construct is designed such that the expression of the selectable marker gene is regulated in such a way. More specifically, transfected cells are screened for expression of the neo gene, which (1) successfully electroporates the cells and (2) inhibits lac repressor inhibition of neo transcription. It needs to be removed by homologous recombination. This method allows the identification of transfected cells and homologous recombinants to be performed in one step by adding a single drug.
[0077]
Alternatively, positive and negative selection techniques can be used to select for homologous recombinants. This technique involves a process in which a first drug (eg, a neomycin-like drug) is added to a cell population to select for growth of transfected cells (ie, positive selection). A second drug (eg, FIAU) is then added to kill cells expressing the negative selection marker (ie, negative selection). Cells that contain and express the negative selectable marker are killed by the selection agent, whereas cells that do not contain and do not express the negative selectable marker survive. For example, cells into which this construct has been inserted heterologously express HSV thymidine kinase, and are therefore expressed in the form of a herpes drug such as ganciclovir (GANC) or FIAU (1- (2-deoxy-2-fluoro). -BD-arabinofuranosyl) -5-iodouracil; 1- (2-deoxy 2-fluoro-BD-arabinofluranosyl) -5-iodouracyl) (e.g. Mansour et al., Nature). 336: 348-352: (1988); Capecchi, Science 244: 1288-1292 (1989); Capecchi, Trends in Genet 5: 70-76 (1989)).
[0078]
Successful recombination can be identified by analyzing the DNA of the selected cells to confirm homologous recombination. Various techniques known in the art (eg, PCR and / or Southern analysis) can be used to confirm a homologous recombination event.
[0079]
Homologous recombination can also be used to disrupt genes in stem cells and other cell types that are not totipotent embryonic stem cells. By way of example, the stem cells can be bone marrow, lymphatic or neural progenitor cells and precursor cells. Such transgenic cells are particularly useful in studying gene function in ontogenetic pathways. Stem cells can be derived from any vertebrate species (eg, mouse, rat, dog, cat, pig, rabbit, human, non-human primate, etc.).
[0080]
In cells that are not totipotent, it may be desirable to knock out both copies of the target using methods known in the art. For example, cells containing homologous recombination at the target locus (positive selectable markers (eg, Neo ^r ) Selected for expression and screened for non-random integration) can further be selected for multiple copies of the selectable marker gene by exposure to elevated levels of a selection agent (eg, G418). The cells are then analyzed for homozygosity at the target locus. Alternatively, a second construct can be generated with a different positive selectable marker inserted between the two homologous sequences. The two constructs can be introduced into the cells, either sequentially or simultaneously, after which appropriate selection can be made for each positive marker gene. The final cells are screened for homologous recombination of both alleles of the target.
[0081]
(Production of transgenic animals)
The selected cells are then injected into the blastocyst of an animal (eg, a mouse) (or other developmental stage appropriate for the purpose of producing a viable animal (eg, a morula)) and the chimera is (See, e.g., Bradley, A., Teratocarcinomas and Embronic Stem Cells: A Practical Approach, Ed. JJ Robertson, IRL, Oxford, pp. 113-152 (1987)). Alternatively, the selected ES cells can be aggregated with dissociated mouse embryo cells to form an aggregation chimera. The chimeric embryo can then be implanted into a suitable pseudopregnant female Foster animal and the embryo can be born. Chimeric progeny that carry the homologously recombined DNA in germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA. In one embodiment, chimeric progeny mice are used to generate mice with heterozygous disruptions in the gene. The heterozygous transgenic mice can then be mated. It is well known in the art that typically one quarter of the offspring of such matings have a homozygous disruption in the target gene.
[0082]
Heterozygous and homozygous transgenic mice can then be compared to normal wild-type mice to determine if disruption of the target gene causes a phenotypic change, particularly a pathological change. For example, heterozygous and homozygous mice can be evaluated for phenotypic changes by physical examination, necropsy, histology, clinical chemistry, complete blood count, body weight, organ weight, and cytological evaluation of bone marrow.
[0083]
In one embodiment, the phenotype (or phenotypic change) associated with the disruption of the target gene is entered or stored in a database. Preferably, the database includes (i) genotype data (eg, identification of a disrupted gene) and (ii) phenotypic data associated with the genotype data (eg, a phenotype resulting from gene disruption). This database is preferably an electronic database. Further, the database is preferably combined with a search tool such that the database is searchable.
[0084]
(Conditional transgenic animal)
The present invention further contemplates conditional transgenic or knockout animals, such as conditional transgenic or knockout animals produced using recombinant methods. Bacteriophage P1 Cre recombinase and flp recombinase from yeast plasmids are two non-limiting examples of site-specific DNA recombinase enzymes, which are specific target sites (the lox P site for cre recombinase, The DNA is then cleaved at the flt site (in a flp recombinase) and catalyzes ligation of this DNA to a second cleavage site. A number of other suitable site-specific recombinases have been described, and their genes can be used in accordance with the methods of the present invention. Such recombinases include Int recombinase of bacteriophage lambda (with or without Xis) (Weisberg, R. et al., Lambda II, (Hendrix, R. et al., Eds.), Cold Spring Harbor Press, Cold Spring. Harbor, NY, 211-50 (1983), which is incorporated herein by reference); TpnI and β-lactamase transposon (Mercier et al., J. Bacteriol., 172: 3745-57 (1990)); Tn3 resolvase (Flanagan & Fennewald, J. Molec. Biol., 206: 295-304 (1989); Stark et al., Cell, 58: 779-90 (1989)); (Matsuzaki et al., J.Bacteriol, 172:. 610-18 (1990)); B. subtilis SpoIVC recombinase (Sato et al., J. Bacteriol. 172: 1092-98 (1990)); Flp recombinase (Schwartz & Sadowski, J. Molec. Biol. 205: 647-658 (1989); Parsonol et al. Chem., 265: 4527-33 (1990); Golic & Lindquist, Cell, 59: 499-509 (1989); Amin et al., J. Molec. Biol., 214: 55-72 (1990)); (Glasgow et al., J. Biol. Chem., 264: 10072-82 (1989)); immunoglobulin recombinase (Malynn et al., Cell, 54: 453-460 (1988). ); And Cin recombinase (Haffter & Bickle, EMBO J., 7: 3991-3996 (1988); Hubner et al., J. Molec. Biol., 205: 493-500 (1989)), all herein incorporated by reference. Which is incorporated therein). Such systems include: Echols (J. Biol. Chem. 265: 14697-14700 (1990)); de Villartay (Nature, 335: 170-74 (1988)); Craig, (Ann. Rev. Genet., 22: 77-105 (1988)); Poyart-Salmeron et al., (EMBO J. 8: 2425-33 (1989)); Hunger-Bertling et al., (Mol Cell. Biochem., 92: 107-16 (1990)). And Cregg & Madden (Mol. Gen. Genet., 219: 320-23 (1989)), all of which are incorporated herein by reference.
[0085]
Cre was purified to homogeneity and the reaction with the loxP site has been extensively characterized (Abremski & Hess J. Mol. Biol. 259: 1509-14 (1984), incorporated herein by reference). Incorporated). Cre protein has a molecular weight of 35,000 and is commercially available from New England Nuclear / DuPont. The cre gene, which encodes the Cre protein, has been cloned and expressed (Abremski et al., Cell 32: 1301-111 (1983), incorporated herein by reference). This Cre protein mediates recombination between two loxP sequences which may be on the same or different DNA molecules (Sternberg et al., Cold Spring Harbor Sym. Quant. Biol. 45: 297-309 (1981)). Since the internal spacer sequence of the loxP site is asymmetric, the two loxP sites may be directional with respect to each other (Hoess & Abremski, Proc. Natl. Acad. Sci. USA 81: 1026-29). (1984)). Thus, if two sites on the same DNA molecule are in a directly repeated orientation, Cre will cut out the DNA between the sites (Abremski et al., Cell 32: 1301-11-1 (1983)). However, if the sites are inverted with respect to each other, the DNA between them is not cut out after recombination, but simply inverted. Thus, a circular DNA molecule having two loxP sites in the direct direction is recombined to produce two smaller circles. In contrast, a circular molecule having two loxP sites in the inverted direction simply reverses the DNA sequence adjacent to the loxP site. In addition, recombinase action can result in the interchange of regions distal to the target site when the target is on a separate DNA molecule.
[0086]
Recombinases have important applications for characterizing gene function in knockout models. When the constructs described herein are used to disrupt a target gene, a fusion transcript is produced when the insertion of the positive selectable marker occurs downstream (3 ') of the translation start site of the target gene. obtain. Fusion transcripts can produce some level of protein expression with unknown results. It has been suggested that insertion of a positive selectable marker gene can affect the expression of nearby genes. Since it is not possible to distinguish whether a given phenotype is associated with gene inactivation or transcription of a nearby gene, these effects indicate that gene function is determined after a knockout event. It can be difficult. Both potential problems are solved by utilizing recombinase activity. If the recombinase site in the same direction is adjacent to the positive selectable marker, the addition of the corresponding recombinase will remove the positive selectable marker. In this way, the effects caused by the expression of the positive selectable marker or the fusion transcript are avoided.
[0087]
In one embodiment, the purified recombinase enzyme is provided to the cells by direct microinjection. In another embodiment, the recombinase is expressed from a co-transfected construct or vector in which the recombinase gene is operably linked to a functional promoter. A further aspect of this embodiment is the use of a tissue-specific or inducible recombinase construct that allows one to select when and where recombination occurs. One method for performing inducible forms of recombinase-mediated recombination involves the use of a vector that expresses the desired recombinase activity using an inducible or tissue-specific promoter or other gene regulatory element. The inducible expression element is preferably operatively arranged to allow for inducible control or activation of expression of the desired recombinase activity. Examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionein, ecdysone, and other steroid responsive promoters, rapamycin responsive promoter, and the like (No et al., Proc. Natl. Acad. Sci. USA, 93: 3346-51 (1996); Furth et al., Proc. Natl. Acad. Sci. USA, 91: 9302-6 (1994)). Additional control elements that can be used include promoters that require specific transcription factors, such as viral promoters. A vector incorporating such a promoter expresses only recombinase activity in a cell that expresses a necessary transcription factor.
[0088]
(Disease model)
The cell and animal based systems described herein can be used as models for disease. Any species of animal, including but not limited to mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats and non-human primates, such as baboons, monkeys, and chimpanzees, can be used as animal models for disease. Can be used to create. In addition, cells obtained from humans can be used. These systems can be used in various applications. Such assays can be used as part of a screening strategy designed to identify agents, such as compounds capable of ameliorating disease symptoms. Thus, animal systems and cell-based system models can be used to identify drugs, medicaments, treatments and interventions that may be effective in treating disease.
Cell-based systems can be used to identify compounds that can act to ameliorate disease symptoms. For example, such cell lines can be exposed to compounds suspected of exhibiting the ability to ameliorate disease symptoms. This is done at a concentration and for a time sufficient to induce such recovery of disease symptoms in the exposed cells. After exposure, the cells are tested to determine whether one or more diseased cellular phenotypes have been altered to resemble a more normal or wild-type non-disease phenotype. You.
In addition, animal-based disease systems such as those described herein can be used to identify compounds that can ameliorate disease symptoms. Such animal models can be used as test substrates to identify drugs, medicaments, treatments and interventions that may be effective in treating the disease or other phenotypic features of the animal. For example, an animal model can be exposed to a compound or agent suspected of exhibiting the ability to ameliorate disease symptoms. This is done at a concentration and for a time sufficient to induce such recovery of disease symptoms in the exposed animal. The response of the animal to the exposure can be monitored by assessing the reversal of the disorder associated with the disease. Exposure can include treating the mother animal during pregnancy of the model animals described herein, thereby exposing the embryo or fetus to a compound or agent that can prevent or ameliorate the disease or phenotype. Newborn, young and adult animals can also be exposed.
[0089]
More specifically, a method of identifying an agent (including a compound) using the animal model of the present invention (specifically, a transgenic mouse) preferably comprises at least a method associated with disruption in a target gene. Provided based on the ability to affect one phenotype. In one embodiment, the invention provides a method of identifying an agent that has an effect on the expression or function of a target gene or, alternatively, a target protein. The method includes, for example, measuring the physiological response of an animal to a drug, and comparing the physiological response of such an animal to a control animal, wherein the target is compared to a control animal. The physiological response of the animal, including the disruption in the gene, indicates the specificity of the drug. "Physiological response" is any biological or physical parameter of an animal that can be measured. To assess physiological responses, molecular assays (eg, gene transcription, protein production and degradation rates), physical parameters (eg, exercise physiology tests, measurement of various parameters of respiration, measurement of heart rate or blood pressure, Measurement of bleeding time, PTT.T, or TT) and cell assays (eg, immunohistochemical assays for cell surface markers, or the ability of cells to aggregate or proliferate) can be used. The transgenic animals and cells of the invention can be used as a model for a disease, disorder or condition associated with a phenotype associated with disruption of a target gene.
[0090]
The present invention provides specialized animal models for testing and developing novel treatments associated with behavioral phenotypes. Behavioral phenotypic analysis can be used, for example, to test the effectiveness of proposed genetic and pharmacological treatments for genetic disorders in humans, such as neurological, neuropsychological, or psychotic disorders. Enables the development of useful animal models.
[0091]
Statistical analysis of the various behaviors measured can be performed using any conventional statistical program routinely used by those skilled in the art, such as "Analysis of Variance" or ANOVA. Although "p" values of about 0.05 or less are generally considered to be statistically significant, slightly higher p values may still indicate a statistically significant difference. To statistically analyze abnormal behavior, a comparison between the behavior of a transgenic animal (or group) and that of a wild-type mouse (or group) is typically performed under certain defined conditions. Performed below. As used herein, “abnormal behavior” refers to an animal having a disruption in a target gene (eg, a transgenic animal) that is different from an animal having no disruption in the target gene (eg, a wild-type mouse). Refers to the behavior shown. Anomalous behavior consists of any number of standard behaviors that can be objectively measured (or observed) and compared. In comparison, it is preferred that the change is statistically significant to confirm that there is really a significant difference in behavior between the knockout animal and the wild-type control animal. Examples of behavior that can be measured or observed include ataxia, rapid limb movement, eye movement, respiration, motor activity, cognition, emotional behavior, social behavior, hyperactivity, irritability, anxiety, learning disability, abnormal rewards Includes, but is not limited to, abnormal social interactions such as behavior and aggression.
[0092]
A series of tests can be used to measure the behavioral phenotype of the animal models of the present invention, including neurological and neuropsychological tests to identify abnormal behavior. These tests can be used, for example, to measure abnormal behaviors related to learning and memory, diet, pain, aggression, sexual reproduction, anxiety, depression, schizophrenia, and substance abuse (eg, Crawley and Paylor) , Hormones and Behavior 31: 197-211 (1997)).
[0093]
Social interaction testing involves exposing mice to other animals in various settings. The animal's social behavior (eg, contact, climbing, smelling and copulation) is subsequently assessed. Differences in behavior can then be analyzed statistically and compared (eg, SE File et al., Pharmacol. Bioch. Behav. 22: 941-944 (1985); RR Holson, Phys. Behav. 37: 239-247 (1986)). The following are examples of the behavior test.
[0094]
The mouse startle response test typically involves exposing the animal to a sensory (typically auditory) stimulus and measuring the animal's startle response (eg, MA Geyer et al., Brain Res). Bull.25: 485-498 (1990); Paylor and Crawley, Psychopharmacology 132: 169-180 (1997)). The pre-pulse suppression test, where percent inhibition (from a normal startle response) is measured by first "cueing" the animal using a short, mild pre-pulse preceding the startle pulse, Can be used.
[0095]
The electric shock test usually involves exposure to a live surface and subsequent measurement of behavior, such as, for example, motor activity, learning, social behavior, and the like. Behavior is measured and analyzed statistically using standard statistical tests (eg, GJ Kant et al., Pharm. Bioch. Behav. 20: 793-797 (1984); NJ Leidenheimer). Et al., Pharmacol. Bioch. Behav. 30: 351-355 (1988)).
[0096]
A tail pinch or immobilization test involves applying pressure to the animal's tail and / or restricting the animal's movement. Motor activity, social behavior, and cognitive behavior are examples of areas that are measured (see, for example, M. Bertolucci D'Angic et al., Neurochem. 55: 1208-1214 (1990)).
[0097]
New tests usually involve exposure to new environments and / or new subjects. The motor behavior of the animal in the new environment and / or around the new subject is measured and analyzed statistically (for example, DK Reinstein et al., Pharm. Bioch. Behav. 17: 193-202 ( 1982); B. Poucet, Behav.Neurosci. 103: 1009-10016 (1989); RR Holson et al., Phys. Behav.37: 231-238 (1986)). This test can be used to detect deficiencies or deficiencies in visual processing.
[0098]
Learned helplessness tests include exposure to stress, such as noxious stimuli, that cannot be affected by animal behavior. Animal behavior can be analyzed statistically using a variety of standard statistical tests (see, for example, A. Leshner et al., Behav. Neural Biol. 26: 497-501 (1979)).
[0099]
Alternatively, a tail suspension test that measures the "immobile" time of a mouse when suspended "upside down" by the tail may be used. This is a measure of whether an animal struggles or not, that is, an indicator of depression. In humans, depression is thought to arise from feelings of lack of control of its life or situation. It is believed that depression can be induced in an animal by repeatedly subjecting the animal to an uncontrolled reversal. Eventually, a "learned helpless" situation is achieved, in which the animal ceases to change its environment and simply accepts its fate. Animals that stop writting earlier are likely to be more depressed. Studies have shown that the administration of certain antidepressants prior to testing increases the time it takes for animals to give up before giving up.
[0100]
The Morris water-maze test involves learning spatial orientation in water and subsequently measuring animal behavior, such as by calculating the number of incorrect choices. The measured behavior is analyzed statistically using standard statistical tests (see, for example, EM Spruijt et al., Brain Res. 527: 192-197 (1990)).
[0101]
Alternatively, a Y-shaped maze can be used (e.g., McFarland, DJ, Pharmacology, Biochemistry and Behavior 32: 723-726 (1989); Dellu, F. et al., Neurobiology of Long-earning: 31-year-old long-moring: (2000)). The Y maze is generally considered a test of cognitive ability. The size of each arm of the Y-maze can be, for example, about 40 cm x 8 cm x 20 cm, although other sizes can be used. Each arm can also have 16 equally spaced photo beams, for example, to automatically detect movement within the arm. At least two different tests can be performed using such a Y maze. In the continuous Y maze paradigm, a mouse is made to search all three arms of the Y maze, for example, for about 10 minutes. Animals are tracked continuously using a photobeam detection grid and the data can be used to measure spontaneous alternation and positive biasing behavior. Spontaneous alternation refers to the natural tendency of "normal" animals to visit the least familiar arm of the maze. Alternations are recorded when the animal makes two consecutive rotations in the same direction, and thus represent successive visits to the maze arm that entered the least frequently. Positional bias measures the tendency of an animal to favor rotation in one direction over the other to determine an eccentrically defined response. Thus, this test may detect differences in an animal's ability to proceed based on an allocentric or egocentric mechanism. Two experimental Y-maze memory tests measure response to novelty and spatial memory based on the free choice search paradigm. During the first test (acquisition), the animal is free to visit the two arms of the Y-maze, for example for about 15 minutes. The third arm is shielded during this test. The second test (recovery) is performed after a test interval of, for example, about 2 hours. During the recovery test, the shielded arm is released and the animal can approach all three arms, for example, for about 5 minutes. Data is collected during recovery studies and analyzed for the number and time of visits to each arm. Since the three arms of the maze are nearly identical, the distinction between novelty and familiarity depends on the "environmental" spatial cues around the room associated with the location of each arm. Changes in admission to a transgenic animal model arm and the duration spent in the new arm can indicate novelty and the role of that gene in mediating the cognitive process.
[0102]
Passive avoidance or shuttle box tests generally involve exposure to more than one environment, one of which is harmful and results in an animal-learned choice. Behavioral measures include, for example, response latency, exact number of responses, and response consistency (eg, R. Ader et al., Psychon. Sci. 26: 125-128 (1972); RR Holson, Phys. Behav. 37: 221-230 (1986)). Alternatively, a zero maze may be used. In the zero maze, the animal is placed, for example, in a closed quadrant of an elevated annular platform having two open quadrants and two closed quadrants and allowed to explore for approximately 5 minutes. This paradigm exploits the approach-avoidance conflict between normal rodent exploratory activity and aversion to open space. This test measures the level of anxiety and can be used to evaluate the effectiveness of anxiolytics. The amount of time spent in the open quadrant versus the closed quadrant can be automatically recorded, for example, by placing a photobeam at each transition location.
[0103]
Food avoidance tests include exposure to fresh food and, for example, objective measurement of food intake and the latency of ingestion. Measured behavior is analyzed statistically using standard statistical tests (see, eg, BA Campbell et al., J. Comp. Physiol. Psychol. 67: 15-22 (1969)). ).
[0104]
The elevated plus maze test involves exposing a sideless maze on the platform, and the behavior of the animal is objectively measured by counting the number of maze intrusions and maze learning. This behavior is analyzed statistically using standard statistical tests (see, for example, HA Baldwin et al., Brain Res. Bull, 20: 603-606 (1988)).
[0105]
The stimulus-induced hyperactivity test involves injection of a stimulant drug (eg, amphetamine, cocaine, PCP, etc.) and objectively measuring, for example, motor activity, social interaction, cognitive behavior. Animal behavior is analyzed statistically using standard statistical tests (eg, PBS Clarke et al., Psychopharmacology 96: 511-520 (1988); P. Kuczenski et al., J. Neuroscience 11). : 2703-2712 (1991)).
[0106]
Self-stimulation tests generally involve giving mice the opportunity to modulate electrical and / or chemical stimulation of the mouse's own brain. Behavior is measured by the frequency and pattern of self-stimulation. Such behavior is statistically analyzed by standard statistical tests (eg, S. Nassif et al., Brain Res., 332: 247-257 (1985); WL Isaac et al., Behav. Neurosci. 103). : 345-355 (1989)).
[0107]
Reward tests embody a variety of behaviors (e.g., motor, cognitive, and social behaviors), e.g., measure the rate and reliability of behavioral changes, and statistically analyze the measured behavior. (See, for example, LE Jarrard et al., Exp. Brain Res. 61: 519-530 (1986)).
[0108]
DRL (differential reinforcement to low rates of response) performance testing involves exposing to an intermittent reward paradigm and measuring the number of appropriate responses (eg, lever presses). Such behavior is analyzed statistically using standard statistical tests (eg, JD Sinden et al., Behav. Neurosci. 100: 320-329 (1986); V. Nalwa et al., Behav. Brain Res. 17: 73-76 (1985); and AJ Nonneman et al., J. Comp. Physiol. Psych. 95: 588-602 (1981)).
[0109]
Spatial learning tests involve exposing to a complex and novel environment, measuring the speed and extent of spatial learning, and statistically analyzing the measured behavior (see, eg, N. Pitsikas et al., Pharm. Bioch. 38: 931-934 (1991); B. Poucet et al., Brain Res. 37: 269-280 (1990); D. Christie et al., Brain Res. 37: 263-268 (1990); and F. Van Haaren. Et al., Behav. Neurosci. 102: 481-488 (1988)). Alternatively, an open field (of) test, where a greater distance moving at a given time is a measure of the animal's activity level and anxiety, may be used. If the open field is a novel environment, it is believed that this open field creates an approach-avoidance situation that becomes a "scissors" between the desire for the animal to seek and the desire to protect itself. It is anticipated that the "normal" mouse will spend more time in the corners and perimeters than in the center, where there is no hiding place, because the room is lightened and there is no hiding place outside the corner. However, "normal" mice go out into the central area as they increasingly explore the room. Then, particularly anxious mice spend most of their time in the corner with relatively little or no exploration of the central area, while bold (ie, non-anxious) mice It can be inferred that the distance moves, indicating that the periphery is less preferred for the central region.
[0110]
Visual, somatosensory and auditory agnosia tests generally expose to sensory stimuli, objectively measure, for example, orientating responses to the environment, and statistically analyze the measured behavior. (See, for example, JM Vargo et al., Exp. Neurol. 102: 199-209 (1988)).
[0111]
Completion behavior testing generally involves feeding and drinking and objectively measuring consumption. Measured behavior is analyzed statistically using standard statistical tests (eg, PJ Fletcher et al., Psychopharmacol. 102: 301-308 (1990); MG Corda et al., Proc. Nat'l.Acad.Sci.USA 80: 2072-2076 (1983)).
[0112]
Visual discrimination tests can also be used to assess visual processing in animals. One or two similar objects are placed in the open field and the animals are allowed to explore for about 5-10 minutes. The time spent searching for each object is recorded (approach to the object, i.e., movement within, for example, about 3-5 cm is considered an object search). The animal is then removed from the open field and the objects are replaced with similar and novel objects. The animal is returned to the open field and the percentage of time spent searching for a novel object versus an old object is measured (again, over a time period of about 5-10 minutes). "Normal" animals typically spend a high percentage of their time searching for novel objects rather than old objects. If a delay is imposed between sampling and testing, the memory task becomes more hippocampal dependent. If no delay is imposed, the task is largely based on simple visual identification. This test can also be used for olfactory discrimination, where objects (preferably simple blocks) can be sprayed or otherwise processed to retain odor. This test can also be used to determine whether an animal is taste discriminable, and animals that return to previously eaten food instead of novel food exhibit taste neophobia.
[0113]
A hot plate analgesia test can be used to assess the sensitivity of an animal to heat or painful stimuli. For example, the mouse can be placed on a hot plate at about 55 ° C., and the response latency of the mouse (eg, the time between lifting the hind paws and licking the hind paws) can be recorded. These responses are not reflexes, but rather "advanced" responses that require cortical involvement. This test can be used to assess nociceptive disorders.
[0114]
The accelerated rotarod test can be used to measure mouse coordination and balance. The animal can be placed on a rod that operates, for example, as a rotating treadmill (or rotating log). The rotarod may rotate slowly at first, and then progressively faster until a speed of, for example, about 60 rpm is reached. The mouse must continuously change its position to avoid falling. Animals are preferably tested at least three times with a minimum of 20 minutes. Mice that can stay on the rod the longest are considered to have better coordination and balance.
[0115]
The metrazole dosing test can be used for screening animals to change their susceptibility to seizures or similar events. For example, a 5 mg / ml solution of methazole can be infused through the tail vein of a mouse, for example, at a rate of about 0.375 ml / min. This injection causes all mice to undergo seizures and subsequently causes lethality. Mice that enter the seizure phase earliest are more likely to have seizures. Four different physiological stages can be recorded: shortly after the start of the infusion, the mice show a marked “convulsion”, followed by a series of seizures, the final tone of the body, known as “tonic extension”. And then death.
[0116]
(Target gene product)
The present invention further contemplates the use of the target gene sequence to produce a target gene product. Target gene products may include proteins that exhibit functionally equivalent gene products. Such equivalent gene products may include deletions, additions or substitutions of amino acid residues within the amino acid sequence encoded by the gene sequences described herein, but which result in silent changes and, thus, Produce a functionally equivalent target gene product. Amino acid substitutions can be made based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved.
[0117]
For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, Positively charged (basic) amino acids include arginine, lysine and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. As used herein, "functionally equivalent" refers to a protein that can exhibit an activity in vivo substantially similar to the endogenous gene product encoded by the target gene sequence. Alternatively, when used as part of an assay, "functionally equivalent" refers to other intracellular molecules or extracellular molecules in a manner substantially similar to that performed by the corresponding portion of the endogenous gene product. A peptide capable of interacting with a molecule.
[0118]
Other protein products useful according to the method of the invention are peptides derived from or based on target genes produced by recombinant or synthetic means (derived peptides).
[0119]
The target gene product may be produced by recombinant DNA technology, using techniques well known in the art. Accordingly, methods for preparing the gene polypeptides and peptides of the invention by expressing a nucleic acid encoding a gene sequence are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing the protein-coding gene sequence and appropriate transcription / translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination (see, eg, Sambrook et al., 1989 (supra) and Ausubel et al., 1989 (supra)). . Alternatively, RNA that can encode the gene sequence of a protein is, for example, an automatic synthesizer (see, for example, Oligonucleotide Synthesis: A Practical Approach, edited by Gait, MJ, IRL Press, Oxford (1984)). Can be chemically synthesized.
[0120]
Various host expression vector systems can be used to express the gene coding sequences of the present invention. Such host expression systems show vehicles that produce the coding sequence of interest and can be subsequently purified, but which, when transformed or transfected with the appropriate nucleotide coding sequence, allow the protein of the gene of the invention to be transformed in situ. The cells that can be shown are also shown. These include microorganisms such as bacteria (eg, E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the protein coding gene sequence; Yeast (eg, Saccharomyces, Pichia) transformed with a recombinant yeast expression vector containing; an insect cell line infected with a recombinant virus expression vector (eg, baculovirus) containing a protein coding gene sequence; a recombinant virus expression vector (Eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with a recombinant plasmid expression vector (eg, a Ti plasmid) containing a protein-encoding gene sequence. Transgenic plant cell lines; or recombinant expression comprising a promoter derived from the genome of a mammalian cell (eg, a metallothionein promoter) or a promoter derived from a mammalian virus (eg, an adenovirus late promoter; vaccinia virus 7.5K promoter). Mammalian cell lines with the construct (eg, COS, CHO, BHK, 293, 3T3) include, but are not limited to.
[0121]
In bacterial systems, many expression vectors may be advantageously selected depending on the use intended for the protein of the gene to be expressed. For example, if large quantities of such proteins are to be produced, vectors that direct the expression of high levels of fusion protein products that are readily purified, e.g., for the production of antibodies or screening of peptide libraries, may be used. It may be desirable. Such vectors include E. coli. coli expression vector pUR278, in which the protein-coding gene sequence can be individually ligated in frame with the lacZ coding region to produce a fusion protein (Ruther et al., EMBO J., 2: 1791-94 (1983)); pIN vector (Inouye & Inouye, Nucleic Acids Res., 13: 3101-09 (1985); Van Heake et al., J. Biol. Chem., 264: 5503-9 (1989)) and the like. But not limited thereto. The pGEX vector can also be used to express a foreign polypeptide as a fusion protein using glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site, so that the cloned target gene protein can be released from the GST moiety.
[0122]
In a preferred embodiment, the full-length cDNA sequence is in-frame at the amino terminus using standard PCR methodology (Innis et al. (Eds.), PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990)). A BamHI site and an in-frame EcoRI site at the carboxyl terminus are added and ligated into the pGEX-2TK vector (Pharmacia, Uppsala, Sweden). The resulting cDNA construct contains a kinase recognition site at the amino terminus for radiolabeling and a glutathione S-transferase sequence at the carboxy terminus for affinity purification (Nilsson et al., EMBO J., 4: 1075-80). (1985); Zabeau et al., EMBO J., 1: 1217-24 (1982)).
[0123]
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. This virus grows in Spodoptera frugiperda cells. Gene coding sequences can be cloned individually into non-essential regions of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter). Successful insertion of the genetic coding sequence results in inactivation of the polyhedrin gene and production of a non-closing recombinant virus (ie, a virus that lacks the proteinaceous coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (eg, Smith et al., J. Virol. 46: 584-93 (1983); U.S. Pat. 4,745,051).
[0124]
In mammalian host cells, a number of viral-based expression systems may be used. In cases where an adenovirus is used as an expression vector, the gene encoding the sequence of interest may be ligated to an adenovirus transcription / translation control complex (eg, the late promoter and tripartite leader sequence). This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (eg, region E1 or region E3) results in a recombinant virus that can survive in the infected host and express the genetic protein (eg, Logan). Et al., Proc. Natl. Acad. Sci. USA 81: 3655-59 (1984)). Certain initiation signals may also be required for efficient translation of the inserted gene coding sequence. These signals include the ATG start codon and adjacent sequences. If the entire gene (including its own initiation codon and adjacent sequences) is inserted into the appropriate expression vector, no additional translational control signals may be required. However, if only a portion of the gene coding sequence is inserted, an exogenous translational control signal (possibly including the ATG start codon) must be provided. Furthermore, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins (both natural and synthetic). The efficiency of expression can be increased by inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (Bitter et al., Methods in Enzymol., 153: 516-44 (1987)).
[0125]
In addition, a host cell type may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. For this purpose, eukaryotic host cells that have the machinery for proper processing of the primary transcript, glycosylation of the gene product, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and the like.
[0126]
It is preferable to produce a recombinant protein with high yield over a long period of time and to stably express the protein. For example, cell lines that stably express the gene protein can be engineered. Rather than using an expression vector that contains the viral origin of replication, the host cell may have DNA controlled by appropriate expression control elements (eg, promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers. Can be used for transformation. Following the introduction of the foreign DNA, the engineered cells can be allowed to grow for 1-2 days in an enriched media, and then the enriched media is replaced with a selective media. The selectable marker of the recombinant plasmid confers resistance to selection and allows the cell to stably integrate the plasmid into its chromosome and proliferate and form foci. The lesion can then be cloned and expanded into a cell line. This method can be advantageously used to engineer cells that express the gene protein. Such engineered cell lines can be particularly useful for screening and evaluating compounds that produce the endogenous activity of the genetic protein.
[0127]
In a preferred embodiment, control of the timing and / or amount of expression of the recombinant protein can be controlled using an inducible expression construct. Inducible constructs and systems for inducible expression of recombinant proteins are well known to those skilled in the art. Examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionein, ecdysone, and other steroid responsive promoters, rapamycin responsive promoter, and the like (No et al., Proc. Natl.Acad.Sci.USA, 93: 3346-51 (1996); Furth et al., Proc.Natl.Acad.Sci.USA, 91: 9302-6 (1994)). Additional control elements that can be used include promoters required for certain transcription factors, such as viral promoters, certain HIV promoters, and the like. In one embodiment, a Tet-inducible gene expression system is used (Gossen et al., Proc. Natl. Acad. Sci. USA, 89: 5547-51 (1992); Gossen et al., Science, 268: 1766-69 (1995). )). The Tet expression system is E. coli. It is based on two regulatory elements from the tetracycline resistance operon of the E. coli Tn10 transposon: a tetracycline repressor protein (TetR) and a tetracycline operator sequence (tetO) that binds to TetR. Using such a system, the expression of the recombinant protein is placed under the control of the tetO operator sequence and is transfected or transformed into a host cell. In the presence of TetR, which is co-transfected into the host cell, expression of the recombinant protein is suppressed due to binding of the TetR protein to the tetO regulatory element. Next, high-level expression of the regulated gene is induced in response to changes in the concentration of tetracycline (Tc) or a Tc derivative (eg, deoxycycline (Dox)) that compete with the tetO element for binding to TetR. obtain. Constructs and materials for expression of the tet-induced gene are available from CLONTECH Laboratories, Inc. , Palo Alto, CA.
[0128]
When used as a component of an assay system, the gene protein may be labeled, either directly or indirectly, to facilitate detection of the complex formed between the gene protein and the test substance. . Any of a variety of suitable labeling systems may be used, including radioisotopes such as 125I; enzyme labeling systems that generate a detectable calorimetric signal or light when exposed to a substance; and fluorescent labels. But not limited thereto. When using recombinant DNA technology to produce a genetic protein for such an assay system, it may be advantageous to engineer the fusion protein, which may facilitate labeling, immobilization, and / or detection.
[0129]
Indirect labeling involves the use of proteins (eg, labeled antibodies) that specifically bind to the gene product. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
[0130]
(Generation of antibodies)
Described herein are methods for the generation of antibodies that can specifically recognize one or more epitopes. Such antibodies include polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') 2 fragments, fragments generated by Fab expression libraries, anti-idio, Type (anti-Id) antibodies and any of the above epitope binding fragments can be, but are not limited to. Such antibodies can be used, for example, in detecting a target gene in a biological sample or as a method for inhibiting aberrant target gene activity. Thus, such antibodies can be used as part of a method of treating a disease and / or by being used as part of a diagnostic technique, a patient can have abnormal levels of a target gene protein, or such Can be tested for the presence of abnormal forms of any protein.
[0131]
For the production of antibodies, various host animals can be immunized by injecting the target gene, its expression product, or a portion thereof. Such host animals may include, but are not limited to, rabbits, mice, and rats, to name a few. Various adjuvants can be used to increase the immunological response depending on the host species. Such adjuvants include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, keyhole limpet hemocyanin, Examples include, but are not limited to, dinitrophenol, and potentially useful human adjuvants, such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
[0132]
Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen (eg, a target gene product or a derivative antigenic functional group thereof). For the production of polyclonal antibodies, a host animal as described above can be immunized by injecting a gene product supplemented with an adjuvant (also described above).
[0133]
Monoclonal antibodies, which are homogeneous populations of antibodies to specific antigens, can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These techniques include hybridoma technology (Kohler and Milstein, Nature, 256: 495-7 (1975); and U.S. Patent No. 4,376,110), human B cell hybridoma technology (Kosber et al., Immunology Today, 4: 72 (1983); Cote et al., Proc. Natl. Acad. Sci. USA, 80: 2026-30 (1983)), and EBV hybridoma technology (Cole et al., Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc. New York, pp. 77-96 (1985)). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Hybridomas producing the mAbs of the invention can be cultured in vitro or in vivo. Generation of high titer mAbs in vivo is currently the preferred method of production.
[0134]
Furthermore, a method developed for the generation of "chimeric antibodies" (by splicing genes from mouse antibody molecules with appropriate antigen specificity together with genes from human antibody molecules with appropriate biological activity) (Morrison et al., Proc. Natl. Acad. Sci., 81: 6851-6855 (1984); Takeda et al., Nature, 314: 452-54 (1985)) can be used. A chimeric antibody is a molecule in which different portions are from different animal species (eg, having a variable region derived from a mouse mAb and a human immunoglobulin constant region).
[0135]
Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778; Bird, Science 242: 423-26 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, And Ward et al., Nature, 334: 544-46 (1989) can be applied to generate gene-single-chain antibodies, which are linked via amino acid cross-linking. Formed by joining the heavy and light chain fragments of the Fv region, resulting in a single chain polypeptide.
[0136]
Antibody fragments that recognize a particular epitope can be generated by known techniques. For example, such fragments include F (ab ') 2 fragments that can be produced by pepsin digestion of antibody molecules, and Fab fragments that can be generated by reducing the disulfide bridges of the F (ab') 2 fragment. However, the present invention is not limited to these. Alternatively, a Fab expression library may be constructed (Huse et al., Science, 246: 127-81 (1989)) to allow for rapid and easy identification of monoclonal Fab fragments with the desired specificity.
[0137]
(Screening method)
The invention may be directed to agonists (ie, factors that bind to and activate the target gene polypeptide) or antagonists (ie, inhibit the activity of the target gene polypeptide or interact with the ligand of the target gene polypeptide). Can be used in the process to screen for important factors. Accordingly, the polypeptides of the present invention may also be used to assess the binding of small molecules and ligands, for example, in cells, cell-free preparations, chimeric libraries, and mixtures of natural products known in the art. Can be used. Any method commonly used to identify and screen for factors that can modulate the receptor can be used according to the present invention.
[0138]
The present invention provides methods for identifying and screening for factors that regulate the expression or function of a target gene. More specifically, cells containing and expressing the target gene sequence can be used to screen for therapeutic agents. Such cells include non-recombinant monocyte cell lines (eg, U937 (ATCC # CRL-1593), THP-1 (ATCC # TIB-202), and P388D1 (ATCC # TIB-63)); endothelial cells (Eg, HUVEC's and bovine aortic endothelial cells (BAEC's)); and common mammalian cell lines (eg, HeLa and COS cells (eg, COS-7 (ATCC # CRL-1651))). May be mentioned. Further, such cells can include recombinant cell lines, transgenic cell lines. For example, the transgenic mice of the invention can be used to generate a cell line containing one or more cell types associated with a disease, which cell line can be used as a cell culture model for the disorder. Although cells, tissues, and primary cultures from the disease transgenic animals of the present invention can be used, generation of continuous cell lines is preferred. For examples of techniques that can be used to derive continuous cell lines from transgenic animals, see Small et al., Mol. Cell. Biol. 5: 642-48 (1985).
[0139]
The target gene sequence can be introduced into the genome of the cell of interest and can be overexpressed in that genome. To overexpress a target gene sequence, the coding portion of the target gene sequence can be linked to a regulatory sequence that can drive gene expression in the cell type of interest. Such regulatory regions are well known to those of skill in the art and can be used without undue experimentation. The target gene sequence may also be disrupted or underexpressed. Cells having a target gene sequence disrupted or underexpressed may be used to screen for factors that may provide, for example, alternative pathways to compensate for any loss of function due to this disruption or underexpression.
[0140]
In vitro systems can be designed to identify compounds that can bind to the target gene product. Such compounds include peptides consisting of amino acids in the D- and / or L-configuration (e.g., in the form of a random peptide library (see e.g., Ram et al., Nature, 354: 82-4 (1991)). )), Phosphopeptides (eg, random or partially degenerate specific phosphopeptide libraries; see, eg, Songyang et al., Cell, 72: 767-78 (1993)), antibodies, and small organic or inorganic molecules. Small molecules include, but are not limited to. The identified compounds may, for example, modulate the activity of a target gene protein, preferably a target gene protein variant; produce a biological function of the target gene protein; or disrupt normal target gene interactions, or It may be useful in screening for compounds that disrupt such interactions.
[0141]
The principle of the assay used to identify compounds that bind to the target gene protein is that a reaction mixture of the target gene protein and the test compound is prepared under conditions and the two compounds are allowed to interact and bind for a sufficient amount of time. And thereby forming complexes that can be removed and / or detected in the reaction mixture. These assays can be performed in various ways. For example, one method for performing such an assay involves anchoring a target gene protein or test substance to a solid phase and detecting the target protein / test substance complex anchored to the solid phase at the end of the reaction. The step of performing In one embodiment of such a method, the target gene protein can be anchored to a solid surface, and the unanchored test compound can be labeled, either directly or indirectly.
[0142]
In practice, microtiter plates are conveniently used. The anchored component may be immobilized by non-covalent or covalent bonds. Non-covalent attachment can be achieved simply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody (preferably, a monoclonal antibody) specific for the protein can be used to anchor the protein to a solid surface. This surface can be prepared in advance and stored.
[0143]
To perform the assay, non-immobilized components are added to the coated surface containing the attached components. After the reaction is complete, unreacted components are removed (eg, washed) under conditions such that any complexes formed remain immobilized on the solid surface. Detection of a complex attached on a solid surface can be achieved in a number of ways. If the previous non-immobilized component has been previously labeled, detection of the label immobilized on the surface indicates that a complex has formed. If the previous non-solidified component has not been previously labeled, an indirect label can be used to detect the complex attached on the surface; for example, the previous non-immobilized component (Specifically, such antibodies can be directly labeled with a labeled anti-Ig antibody or indirectly labeled).
[0144]
Alternatively, the reaction can be performed in the liquid phase, the reaction product is separated from unreacted components, and the complex is any complex formed in solution, e.g., specific for the target gene product or test compound. Is detected using a labeled antibody specific for the immobilized antibody for attaching the conjugate, and other components of the complex potentially for detecting the attached conjugate.
[0145]
Compounds that are shown to bind to a particular labeled gene product by one of the methods described above can be further tested for their ability to elicit a biochemical response from the target gene protein. Agonists, antagonists and / or inhibitors of the expression product can be identified using assays well known in the art.
[0146]
(Antisense, ribozyme, and antibody)
Other agents that can be used as therapeutics include target genes, their expression products and functional fragments thereof. In addition, agents that reduce or inhibit mutated target gene activity can be used to ameliorate disease symptoms. Such agents include antisense, ribozymes and triple helix molecules. Techniques for making and using such molecules are well known to those skilled in the art.
[0147]
Antisense RNA and DNA molecules act to hybridize to target mRNA and block protein translation, thereby directly blocking translation of mRNA. For antisense DNA, preferred are translation initiation sites, for example, oligodeoxyribonucleotides derived between the -10 and +10 regions of the target gene nucleotide sequence of interest.
[0148]
Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. The composition of the ribozyme molecule must include one or more sequences complementary to the target gene mRNA, and must include the well-known catalytic sequence responsible for mRNA cleavage. See US Pat. No. 5,093,246 for this sequence, which is hereby incorporated by reference in its entirety. Engineered hammerhead motif ribozyme molecules that specifically and effectively catalyze endonucleolytic cleavage of an RNA sequence encoding a target gene protein are themselves within the scope of the present invention.
[0149]
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites, including the following sequences, GUA, GUU and GUC. Once identified, a short RNA sequence of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may make the oligonucleotide sequence unsuitable, such as secondary structure. Can be evaluated for structural features. The suitability of candidate sequences can also be assessed by using a ribonuclease protection assay to test their accessibility for hybridization with complementary oligonucleotides.
[0150]
The nucleic acid molecule used for triple helix formation for inhibition of transcription should be single-stranded and composed of deoxyribonucleotides. The base composition of these oligonucleotides must be designed to promote triple helix formation according to Hoogsteen base pair rules. This generally requires a fairly large stretch of either purines or pyrimidines present on one of the duplexes. The nucleotide sequence can be pyrimidine-based, resulting in TAT and CGC triplets across the three related strands of the resulting triple helix. A pyrimidine-rich molecule provides a base that is complementary to a single-stranded purine-rich region of a duplex oriented parallel to the strand. In addition, purine-rich nucleic acid molecules containing, for example, G residue extensions can be selected. These molecules form a triple helix with the GC pair-rich DNA duplex. There, the majority of the purine residues are located on one of the target duplexes, resulting in a GGC triplet across the triplex of the triplex.
[0151]
Alternatively, the potential sequences that can be targeted for triple helix formation can be increased by making so-called "switchback" nucleic acid molecules. The switchback molecule base pairs with one strand of the duplex first, then the other strand, and a significant extension of either the purine or pyrimidine present on one strand of the duplex. Are synthesized in an alternating 5'-3 ', 3'-5' fashion so as to eliminate the need for
[0152]
The antisense, ribozyme and / or triple helix molecules described herein are capable of transcription (triple helix) and / or translation (antisense, ribozyme) of mRNA produced by both normal and mutant target gene alleles. It is possible to reduce or prevent it. To ensure that a sufficiently normal degree of target gene activity is maintained, the nucleic acid molecule encoding and expressing the target gene polypeptide exhibiting normal activity may comprise any antisense, ribozyme, or triplex used. It can be introduced into cells that do not contain sequences that are also sensitive to helical processing. Also, it may be preferable to co-administer a normal target gene protein into the cell or tissue to maintain an essential degree of target gene activity in the cell or tissue.
[0153]
Antisense RNA and DNA, ribozymes, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include the techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art, such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of a DNA sequence encoding an antisense RNA molecule. Such DNA sequences can be introduced into a wide variety of vectors that incorporate a suitable RNA polymerase promoter, such as a T7 or SP6 polymerase promoter. Also, depending on the promoter used, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be stably introduced into cell lines.
[0154]
Various well-known modifications of DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include the addition of flanking sequences of ribonucleotides or deoxyribonucleotides at the 5 'and / or 3' end of the molecule, or phosphorothioate or 2'O rather than phosphodiesterase linkages in the backbone of oligodeoxyribonucleotides. -Methyl, but is not limited thereto.
[0155]
Antibodies that are specific for both the target gene protein, and particularly the mutant gene protein, and that interfere with its activity, can be used to block mutant target gene function. Such antibodies are known to the protein itself or to a peptide corresponding to a portion of the protein, using standard techniques known in the art and as described herein. Can be generated. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, Fab fragments, single chain antibodies, chimeric antibodies, and the like.
[0156]
In instances where the target gene protein is intracellular and whole antibodies are used, it may be preferable to internalize the antibodies. However, lipofectin liposomes can be used to deliver antibodies or fragments of the Fab region that bind to a target gene epitope into cells. If fragments of an antibody are used, the smallest inhibitory fragment that binds to the binding domain of the target or spread target protein is preferred. For example, a peptide having an amino acid sequence corresponding to the domain of the variable region of the antibody that binds to the target gene protein can be used. Such peptides can be chemically synthesized or produced by recombinant DNA technology using methods well known in the art (eg, Creighton, Proteins: Structures and Molecular Principles (1984) W H. Freeman, New York 1983, supra; and Sambrook et al., 1989, supra). Also, a single-chain neutralizing antibody that binds to an intracellular target gene epitope can be administered. Such single chain antibodies are described, for example, in Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-93 (1993), and the like, for example, by expressing a nucleotide sequence encoding a single-chain antibody in the target cell population.
[0157]
The RNA sequence encoding the target gene protein can be administered directly to a patient exhibiting a disease condition at a concentration that is sufficient for the production of the target gene protein to a degree such that the disease condition is ameliorated. Patients may be treated with gene replacement therapy. One or more copies of a normal target gene, or a portion of a gene that directs the production of a normal target gene protein with target gene function, can be inserted into cells using a vector. Vectors include, but are not limited to, adenovirus, adeno-associated virus, and retroviral vectors, as well as other particles that introduce DNA into cells, such as liposomes. In addition, techniques such as those described above can be used to introduce normal target gene sequences into human cells.
[0158]
The cells containing the gene sequence expressing the normal target gene, preferably autologous cells, can then be introduced or re-introduced into the patient at a location that allows for amelioration of the disease symptoms.
[0159]
(Pharmaceutical composition, effective dosage, and route of administration)
Identified compounds that inhibit the expression, synthesis and / or activity of the target mutant gene can be administered to a patient at a therapeutically effective dose to treat or ameliorate the disease. A therapeutically effective dose refers to that amount of the compound that is sufficient to effect amelioration of the symptoms of the disease.
[0160]
The toxic and therapeutic efficacy of such compounds is e.g. ₅₀ (Lethal dose of 50% of the population) and ED ₅₀ (A therapeutically effective amount of 50% of the population) can be determined by standard pharmaceutical methods of cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and the LD ₅₀ / ED ₅₀ . Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, target sites of diseased tissue may be targeted to such compounds in order to minimize potential damage to uninfected cells and thereby reduce side effects. Care should be taken to design a delivery system that does.
[0161]
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably that of the ED with little or no toxicity. ₅₀ Is within the circulating concentration range. This dosage may vary within this range depending on the dosage form employed and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Dosage is IC as determined in cell culture ₅₀ (I.e., the concentration of the test compound that achieves half-maximal suppression of symptoms) can be formulated in animal models to achieve a circulating plasma concentration range that includes the compound. Such information can be used to more accurately determine useful doses in humans. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.
[0162]
Pharmaceutical compositions for use according to the invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, this compound and its physiologically acceptable salts and solvates may be inhaled or insufflated (either through the mouth or nose) or orally, buccally, parenterally, topically, subcutaneously, intraperitoneally, intravenously. May be formulated for administration by intrapleural, intraocular, intraarterial, or rectal administration. It is also envisioned that the pharmaceutical composition may be administered with other products that enhance the activity of the compound, and may optionally include other therapeutic ingredients.
[0163]
For oral administration, the pharmaceutical composition may comprise, for example, a binder (eg, pregelatinized corn, polyvinylpyrrolidone or hydroxypropylmethylcellulose); a filler (eg, lactose, microcrystalline cellulose or hydrogen phosphate). Pharmaceuticals such as calcium); lubricants (eg, magnesium stearate, talc or silica); disintegrants (eg, potato starch or sodium starch glycolate); or wetting agents (eg, sodium lauryl sulfate). It can take the form of, for example, tablets or capsules prepared by conventional means using commercially acceptable excipients. Tablets may be coated in a manner well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. . Such liquid preparations may include suspending agents (eg, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (eg, lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol or Fractionated vegetable oils); and preservatives (eg, methyl or propyl p-hydroxybenzoate or sorbic acid) and may be prepared by conventional means using pharmaceutically acceptable additives. The preparation may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
[0164]
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
[0165]
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
[0166]
In the case of administration by inhalation, the compounds for use according to the invention may be prepared by pressurization using a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It is conveniently delivered in the form of an aerosol spray from a pack or nebulizer. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules or cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.
[0167]
The compound may be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Good. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.
[0168]
The compound can also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides. Ingestion is probably the easiest method of administration. Such routes of administration are generally simple and straightforward, and often are the least inconvenient or uncomfortable from the patient's point of view. However, this involves passing the substance through the stomach, which is an undesirable environment for many substances, including proteins and other biologically active compositions. The stomach's acidic, hydrolyzable and proteolytic environment effectively promotes the digestion of proteinaceous substances into amino acids and oligopeptides for subsequent assimilation, so that it can be widespread when simply taken orally. It is not surprising that only a few of the diverse biologically active proteinaceous substances survive through the stomach and are absorbed by the body in the small intestine. As a result, many proteinaceous drugs often have to be taken in other ways, such as parenterally by subcutaneous, intramuscular or intravenous injection.
[0169]
Pharmaceutical compositions may also include carriers such as various buffers (eg, Tris, acetate, phosphate), solubilizers (eg, Tween, polysorbate), human serum albumin, etc., to stabilize drug activity. , Preservatives (thimerosal, benzyl alcohol) and antioxidants such as ascorbic acid. The stabilizer can be a surfactant such as Tween-20, Tween-80, NP-40 or Triton X-100. EBP can also be introduced into microparticle preparations of macromolecules for controlled delivery to patients over an extended period of time. A larger search for components in pharmaceutical compositions is available from Remington's Pharmaceutical Sciences, 18th (ed.), R. Gennaro (eds.), Mack Publishing, Easton, Pa. (1990).
[0170]
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (eg, as an emulsifier in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (eg, as sparingly soluble salts). ) Can be prescribed.
[0171]
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
[0172]
(Diagnostics)
Various methods can be used to diagnose disease symptoms associated with the target gene. Specifically, reagents can be used, for example, to detect the presence of a target gene mutation, or to detect either overexpression or underexpression of the target gene mRNA.
[0173]
According to the diagnosis and prognosis method of the present invention, a change in a wild-type target locus is detected. Further, the method can be practiced by detecting a wild-type target locus and identifying a disease predisposition or lack of neoplasia. "Wild-type gene alteration" encompasses all forms of mutation, including deletions, insertions and point mutations in the coding and non-coding regions. Deletions may be of the entire gene or only a portion of the gene. Point mutations can result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations occur only in certain tissues (eg, tumor tissue) and are not inherited in the germline. Germline mutations can be found and inherited in any of the body tissues. When only one allele is mutated in a somatic cell, an early neoplastic state is indicated. However, if both alleles are mutated, a late neoplastic state may be indicated. Thus, the discovery of genetic mutations provides both diagnostic and prognostic information. Target gene alleles that are not deleted (eg, found on sister chromosomes relative to the chromosome with the target gene deletion) can be screened for other mutations such as insertions, small deletions, and point mutations. Mutations found in tumor tissue may be associated with reduced expression of the target gene product. However, mutations that result in a non-functional gene product may also be associated with a cancer condition. Point mutation events can occur in regulatory regions, such as in the promoter of a gene that results in a loss or decrease in expression of mRNA. Point mutations can also result in loss of expression of the target gene product or abolish proper RNA processing resulting in decreased mRNA stability or translation efficiency.
[0174]
One test that can be used to detect mutations in candidate loci is to directly compare genomic target sequences from cancer patients to those from a control population. Also, messenger RNA can be sequenced, for example, after amplification by PCR, thereby eliminating the need to determine the exon structure of a candidate gene. Mutations in cancer patients that fall outside the coding region of the target gene can be detected by testing non-coding regions, such as introns and regulatory sequences near or in the target gene. An early indication that mutations in non-coding regions are important can be found from Northern blot experiments that reveal abnormally large or large numbers of messenger RNA molecules in cancer patients compared to control individuals.
[0175]
The methods described herein can be performed, for example, using a prepackaged diagnostic kit that includes at least one specific gene nucleic acid or anti-gene antibody reagent described herein. This can be conveniently used, for example, in a clinical setting to diagnose patients who show disease symptoms or are at risk of developing the disease.
[0176]
Any cell type or tissue in which the gene is expressed, preferably monocytes, endothelial cells or smooth muscle cells, can be used in the diagnostics described below.
[0177]
DNA or RNA obtained from the cell type or tissue being analyzed can be readily isolated using methods well known in the art. Diagnostic techniques can also be performed directly in situ on tissue sections (fixed and / or frozen) of patient tissue obtained from biopsy or resection so that nucleic acid purification is not required. Nucleic acid reagents can be used as probes and / or primers for such in situ procedures (see, eg, Nuovo, PCR In Situ Hybridization: Protocols and Applications, Raven Press, NY (1992)). .
[0178]
The gene nucleotide sequence, either RNA or DNA, can be used in, for example, hybridization or amplification assays of biological samples to detect disease-related gene structure and expression. Such assays may include, but are not limited to, Southern or Northern analysis, restriction fragment length polymorphism assays, single stranded conformation polymorphism assays, in situ hybridization assays, and polymerase chain reaction assays. Such an analysis can reveal both quantitative aspects of the expression pattern of a gene, as well as qualitative aspects of gene expression and / or gene composition. That is, such aspects may include, for example, point mutations, insertions, deletions, chromosomal rearrangements, and / or activation or inactivation of gene expression.
[0179]
Preferred diagnostic methods for the detection of gene-specific nucleic acid molecules include, for example, one or more labeled nucleic acid reagents and nucleic acids obtained from the cell type or tissue being analyzed to a complementary sequence within the nucleic acid molecule of interest. Contacting and incubating under conditions favorable for specific annealing of these reagents. Preferably, these nucleic acid reagents are at least 9 to 30 nucleotides in length. After incubation, all unannealed nucleic acid is removed from the nucleic acid: fingerprint molecule hybrid. The presence of nucleic acids from the hybridized fingerprint tissue is then detected when such molecules are present. Using such a detection scheme, nucleic acids from the tissue or cell type of interest can be deposited on, for example, a solid support (eg, a membrane) or a plastic surface (eg, a surface on a microtiter plate or polystyrene beads). Can be immobilized. In this case, after incubation, the non-annealed labeled nucleic acid reagent is easily removed. Detection of the remaining annealed labeled nucleic acid reagent is accomplished using standard techniques well known to those skilled in the art.
[0180]
Alternative diagnostic methods for the detection of gene-specific nucleic acid molecules include, for example, PCR (experimental embodiment described in Mullis US Pat. No. 4,683,202 (1987)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA, 88: 189-93 (1991)), self sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87: 874-78 (1990)), Transcription amplification system (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86: 1173-77 (1989)), Qβ replicase (Lizardi et al., Bio / Technology, 6: 1197 (1988)), or any other. Increase nucleic acid Their amplification by the method followed, may include the detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very small numbers.
[0181]
In one embodiment of such a detection scheme, a cDNA molecule is obtained from an RNA molecule of interest (eg, by reverse transcription of the RNA molecule into cDNA). Cell types or tissues from which such RNA can be isolated include any tissue in which a wild-type fingerprint gene is known to be expressed, including monocytes, endothelial cells, and / or smooth muscle, It is not limited to these. The sequence in the cDNA is then used as a template for a nucleic acid amplification reaction, such as a PCR amplification reaction. The nucleic acid reagent used as a synthesis initiation reagent (eg, primer) in the reverse transcription and nucleic acid amplification steps of this method can be selected from among the gene nucleic acid reagents described herein. The preferred length of such a nucleic acid reagent is at least 15 to 30 nucleotides. For detection of amplification products, nucleic acid amplification can be performed using radiolabeled or non-radiolabeled nucleotides. Alternatively, a fully amplified product can be produced, so that the product can be visualized by standard ethidium bromide staining or by using any other suitable nucleic acid staining method.
[0182]
Antibodies to wild-type or mutant gene peptides can also be used as disease diagnostics and prognostics. Such diagnostic methods can be used to detect abnormalities in the level of gene protein expression, or in the structure and / or tissue, intracellular, or subcellular location of the fingerprint gene protein. Structural differences can include, for example, differences in the size, electronegativity, or antigenicity of the mutant fingerprint gene protein relative to the normal fingerprint gene protein.
[0183]
Proteins obtained from the tissue or cell type to be analyzed can be readily detected or isolated using techniques well known to those of skill in the art, including but not limited to Western blot analysis. For a detailed description of methods for performing Western blot analysis, see, for example, Sambrook et al., (1989) supra, Chapter 18. Methods for detecting and isolating proteins used herein are also described, for example, in Harlow and Lane (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)). Method.
[0184]
Preferred diagnostic methods for the detection of wild-type or mutant gene peptide molecules can include, for example, immunoassays in which a fingerprint gene peptide is detected by interaction with an anti-fingerprint gene-specific peptide antibody.
[0185]
For example, an antibody, or antibody fragment, useful in the present invention can be used to quantitatively or qualitatively detect the presence of a wild-type or mutant gene peptide. This can be achieved, for example, by immunofluorescence techniques using fluorescently labeled antibodies (see below) in combination with light microscopy detection, flow cytometry detection, or fluorescence detection. Such a technique is particularly preferred when the fingerprint gene peptide is expressed on the cell surface.
[0186]
Antibodies (or fragments thereof) useful in the present invention can further be used histologically, such as in immunofluorescence or immunoelectron microscopy for in situ detection of fingerprint gene peptides. In situ detection can be accomplished by removing a histological specimen from a patient and applying the labeled antibody of the invention to the specimen. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) on a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the fingerprint gene peptide, but also its distribution in the tissue tested. Using the present invention, those of skill in the art will readily appreciate that any of a wide variety of histological methods (eg, staining procedures) can be modified to achieve such in situ detection.
[0187]
Immunoassays for wild-type, mutant or extended fingerprint gene peptides typically involve the detection of biological fluids, tissue extracts in the presence of a detectably labeled antibody capable of identifying the fingerprint gene peptide. Incubating a biological sample, such as freshly harvested cells or cells incubated in tissue culture, and detecting bound antibody by any of a number of techniques well known in the art. Process.
[0188]
The biological sample can be contacted with and immobilized on a solid support or carrier, such as nitrocellulose, or other solid support capable of immobilizing cells, cell particles or soluble proteins. The support can then be washed with a suitable buffer and subsequently treated using a detectably labeled gene-specific antibody. The solid support may then be washed a second time with a buffer to remove unbound antibody. The amount of bound label on the solid support can then be detected by conventional means.
[0189]
The term "solid support or carrier" is intended to include any support capable of binding an antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural or modified cellulose, polyacrylamide, gabbro, magnetite. The nature of the carrier may be either soluble or insoluble for the purposes of the present invention. The support material can have virtually any possible structural arrangement, so long as the bound molecule can bind to the antigen or antibody. Thus, the arrangement of the supports may be spherical, such as beads, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, and the like. Preferred supports include polystyrene beads. One skilled in the art will know many other suitable carriers for binding antibodies or antigens, or will be able to ascertain a suitable carrier using routine experimentation.
[0190]
The binding activity of a given lot of anti-wild-type or anti-mutant fingerprint gene peptide antibodies can be determined according to well-known methods. One of skill in the art can determine the operational and optimal assay conditions for each determination by using routine experimentation.
[0191]
One of the ways in which a gene peptide-specific antibody can be detectably labeled is by linking the antibody to an enzyme and using it in an enzyme immunoassay (EIA) (Voller, Ric Clin Lab, 8: 289-). 98 (1978) [“The Enzyme Linked Immunosorbent Assay (ELISA)”, Diagnostic Horizons 2: 1-7, 1978, Microbiological Associates, Colleges, Inc., Quarterly Publishing. -20 (1978); Butler, Meth. Enzymol., 73: 482-523 (1981); Maggio (eds.). Enzyme Immunoassay, CRC Press, Boca Raton, Fla (1980);. Ishikawa et al. (Eds.), Enzyme Immunoassay, Igaku-Shoin, Tokyo (1981)). The enzyme bound to the antibody can be combined with a suitable substrate (preferably a chromogenic substrate), for example, in a manner that produces a chemical moiety that can be detected by spectrophotometric, fluorometric, or visible means. react. Enzymes that can be used to detectably label the antibody include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate, dehydrogenase, triosephosphate isomerase, horseradish Examples include, but are not limited to, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection can be achieved by a colorimetric method that uses a chromogenic substrate for the enzyme. Detection can also be achieved by visual comparison of the extent of enzymatic reaction of the substrate to similarly prepared standards.
[0192]
Detection can also be achieved using any of a variety of other immunoassays. For example, by radiolabeling an antibody or antibody fragment, it is possible to detect wild type, mutant, or extended peptides of the fingerprint gene through the use of a radioimmunoassay (RIA) (eg, Weintraub, B. , Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Technologies, The Endocrine Society, March, 1986). The radioisotope can be detected by such means as the use of a gamma counter or a scintillation counter, or by autoradiography.
[0193]
It is also possible to label the antibody with a fluorescent compound. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by fluorescence. Among the most commonly used fluorescently labeled compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.
[0194]
Antibodies can also be detectably labeled using a fluorescent metal such as 152Eu, or others of the lanthanide series. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0195]
Antibodies can also be detectably labeled by binding to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts and oxalates.
[0196]
Similarly, bioluminescent compounds can be used to label the antibodies of the invention. Bioluminescence is a type of chemiluminescence found in biological systems where catalytic proteins increase the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.
[0197]
In this application, various publications, patents and published patent applications are referenced by way of identification. The disclosures of these publications, patents and published patent specifications referred to in this application are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
[0198]
The following examples are intended only to illustrate the invention and should not be construed as limiting the invention.
[0199]
(Example)
Example 1 Generation and Analysis of Mice Containing Anaphylatoxin C3a Receptor Gene Disruption
To investigate the role of the anaphylatoxin C3a receptor, disruptions in the anaphylatoxin C3a receptor gene were created by homologous recombination. Specifically, transgenic mice containing a disruption in the anaphylatoxin C3a receptor gene were generated. More specifically, as shown in FIGS. 2A-2B, an anaphylatoxin C3a receptor-specific targeting construct having the ability to disrupt or modify the anaphylatoxin C3a receptor gene (specifically, including SEQ ID NO: 1) is provided. Oligonucleotide sequences identified herein as SEQ ID NO: 3 or SEQ ID NO: 4 were generated using as targeting arms (homologous sequences) in this construct.
[0200]
This targeting construct was introduced into ES cells from the 129 / Sv- + P + Mgf-SLJ / J mouse subline to produce chimeric mice. F1 mice were generated by crossing with C57BL / 6 females, and F2 homozygous mutant mice were generated by crossing male and female F1 heterozygotes.
[0201]
Transgenic mice containing a disruption in the anaphylatoxin C3a receptor gene were analyzed for phenotypic changes and expression patterns. The phenotype associated with the disruption in the nuclear receptor gene was determined. Homozygous mice exhibited at least one of the following phenotypes:
Thymus. Abnormalities in the thymus of mutant mice, including thymus of reduced size and reduced weight compared to wild type mice. In particular, homozygous mice have smaller thymus at necropsy and reduced thymus weight and reduced thymus-to-weight ratio compared to wild-type mice, as shown in FIG. 3 and Table 1 below. Was reported:
[0202]
[Table 1]

Expression. Total RNA was isolated from organs or tissues from adult C57B1 / 6 wild type mice. RNA was DNaseI treated and reverse transcribed using random primers. The resulting cDNA was examined for the absence of genomic contamination using primers specific for non-transcribed genomic mouse DNA. The cDNA was averaged for concentration using HPRT primers. RNA transcripts are found in the cerebrum, cortex, subcortical area, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Halder gland, heart, lung, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow, skin, bladder, and lower Detectable in pituitary, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, testis, epididymis, seminal vesicle, coagulating gland, prostate, ovary, uterus and adipose tissue Met. RNA transcripts were not detectable in the gallbladder.
[0203]
(behavior)
For behavioral studies, homozygous mice were generated as follows:
The targeting construct described above is introduced into ES cells from the 129 / SvEv mouse subline to create chimeric mice. F1N0 mice were generated by crossing with C57BL / 6 females. F2N0 homozygous mutant mice were generated by crossing male and female F1 heterozygotes. F1N0 heterozygotes were backcrossed to C57BL / 6 mice to create F1N1 heterozygotes. F2N1 homozygous mice were generated by crossing male and female F1N1 heterozygotes.
[0204]
Homozygous mice exhibited the following behavioral phenotype:
Homozygous mice required significantly lower doses of metrazol to elicit a characteristic seizure-like response (showing increased sensitivity to seizures). Homozygous mutants also showed significantly reduced PPI with a pre-pulse of 90 dB, indicating a stimulatory deficiency similar to that observed in schizophrenic patients.
[0205]
(Example 2: Generation and analysis of mouse containing 5-HT5A gene disruption)
To investigate the role of the 5-HT5A gene, a disruption in the 5-HT5A gene was generated by homologous recombination. Specifically, transgenic mice containing a disruption in the 5-HT5A gene were generated. More specifically, as shown in FIGS. 5A-5B, a 5-HT5A-specific targeting construct capable of disrupting or modifying the 5-HT5A gene (including SEQ ID NO. An oligonucleotide sequence identified herein as SEQ ID NO: 7 or SEQ ID NO: 8 was generated using this targeting arm (homologous sequence) in this construct.
[0206]
This targeting construct was introduced into ES cells from the 129 / Sv- + P + Mgf-SLJ / J mouse subline to produce chimeric mice. F1 mice were generated by crossing with C57BL / 6 females, and F2 homozygous mutant mice were generated by crossing male and female F1 heterozygotes.
[0207]
Transgenic mice containing a disruption in the 5-HT5A gene were analyzed for phenotypic changes and expression patterns.
[0208]
Expression. LacZ (β-galactosidase) expression was detectable in brain and esophagus. In the brain, staining was limited to the cerebellum with Purkinje cells and the nucleus of the granular layer showing expression. In wholemount staining, lacZ expression was detectable only in the cerebellum. Coronal sections of the cerebellum revealed that the X-Gal signal was restricted to the Purkinje cell layer. In addition, another nucleus adjacent to Purkinje cells showed staining. Strong X-gal signals were also present in squamous epithelial cells.
[0209]
Example 3 Generation and Analysis of Mice Containing Chordin Gene Disruption
To investigate the role of cordin, disruptions in the cordin gene were created by homologous recombination. Specifically, transgenic mice containing a disruption in the cordin gene were generated. More specifically, as shown in FIGS. 5A-5B, a cordin-specific targeting construct capable of disrupting or modifying a cordin gene (including SEQ ID NO: 9 in particular) is herein described. An oligonucleotide sequence identified as SEQ ID NO: 11 or SEQ ID NO: 12 was generated using as a targeting arm (homologous sequence) in this construct.
[0210]
This targeting construct was introduced into ES cells from the 129 / Sv- + P + Mgf-SLJ / J mouse subline to produce chimeric mice. F1 mice were generated by crossing with C57BL / 6 females, and F2 homozygous mutant mice were generated by crossing male and female F1 heterozygotes.
[0211]
Transgenic mice containing a disruption in the cordin gene were analyzed for phenotypic changes and expression patterns. The phenotype associated with the disruption in the cordin gene was determined. Homozygous mice exhibited at least one of the following phenotypes: abnormal pain, reduced response to pain and anxiety.
[0212]
Expression. Total RNA was isolated from organs or tissues from adult C57B1 / 6 wild type mice. RNA was DNaseI treated and reverse transcribed using random primers. The resulting cDNA was examined for the absence of genomic contamination using primers specific for non-transcribed genomic mouse DNA. The cDNA was averaged for concentration using HPRT primers. RNA transcripts are found in the cerebrum, cortex, subcortical area, cerebellum, brain stem, eye, heart, lung, liver, pancreas, kidney, skin, gallbladder, bladder, pituitary, adrenal gland, salivary gland, tongue, stomach, colon, cecum, It was detectable in testis, epididymis, seminal vesicles, coagulation gland, prostate, ovary, and uterus.
[0213]
(behavior)
For behavioral studies, homozygous mice were generated as follows:
The targeting construct described above was introduced into ES cells from the 129 / SvEv mouse subline to generate chimeric mice. F1N0 mice were generated by crossing with C57BL / 6 females. F2N0 homozygous mutant mice were generated by crossing male and female F1 heterozygotes. F1N0 heterozygotes were backcrossed to C57BL / 6 mice to create F1N1 heterozygotes. F2N1 homozygous mice were generated by crossing male and female F1N1 heterozygotes.
[0214]
Homozygous mice exhibited the following behavioral phenotype:
F2N0 homozygous mutant mice showed a statistically significant increase in their response latency to the hot plate test compared to wild type animals. Homozygous mice showed a statistically significant increase in the amount of time to hind foot licking when placed on a 55 ° C. hot plate when compared to F2N0 wild type mice. However, N1 generation animals did not show any differences. This test was used to indicate a noxious disorder (ie, a reduced pain response).
[0215]
Homozygous mutant animals from both the N0 and N1 generations tended to spend more time in the central region of the open field test chamber. This test may indicate an anxiety disorder.
[0216]
(Example 4: Generation and analysis of mouse containing RORγ gene disruption)
To investigate the role of RORγ, disruptions in the RORγ gene were created by homologous recombination. Specifically, transgenic mice containing a disruption in the RORγ gene were generated. More specifically, as shown in FIGS. 9A-9B, a RORγ-specific targeting construct capable of disrupting or modifying the RORγ gene (including SEQ ID NO: 13 in particular) is herein described. The oligonucleotide sequence identified as SEQ ID NO: 15 or SEQ ID NO: 16 was generated using the targeting arm (homologous sequence) in this construct.
[0219]
This targeting construct was introduced into ES cells from the 129 / Sv- + P + Mgf-SLJ / J mouse subline to produce chimeric mice. F1 mice were generated by crossing with C57BL / 6 females, and F2 homozygous mutant mice were generated by crossing male and female F1 heterozygotes.
[0218]
Transgenic mice containing a disruption in the RORγ gene were analyzed for phenotypic changes and expression patterns. The phenotype associated with the disruption in the nuclear receptor gene was determined. Homozygous mice exhibited at least one of the following phenotypes:
Lymph nodes. Lymph nodes were absent in all female homozygous mice. In addition, gastrointestinal-associated lymphoid tissue (GALT) was absent in routine sections of intestine from all homozygous mutants.
[0219]
lymphocytes. Compared to wild-type mice, minimal to moderate lymphocyte accumulation was present in various tissues (liver, lung, pancreas, salivary gland and thyroid) in mutant mice. Homozygous mutant mice had multiple organs with lymphoid invasion consistent with lymphoma. This invasion varies from moderate to severe and can involve any organ system. These cells had an increased mitotic rate and frequent apoptosis. Complications tend to be most severe in the spleen and thymus. Other organs involved include: liver, gallbladder, lung, kidney, bladder, heart, aorta, bone, bone marrow, pituitary, adrenal gland, thymus, brain and spinal cord (meninges), pancreas, stomach, Small and large intestine, larynx, trachea, esophagus, tongue, skeletal muscle, testis, epididymis, bladder, seminal vesicles, eyes and Halder glands.
[0220]
Two clinically ill homozygous females (20774 and 20299) and two clinically ill homozygous males (19922 and 20294) had greatly elevated total lymphocyte prevalence. It had a white blood cell count. In one leukemic female 20774, lymphocytes were immature (blastic). This is consistent with the leukemia stage of lymphoma. Affected mouse homozygotes showed signs of the disease, including poor grooming and reduced function. This mouse had increased thoracic and abdominal dimensions, hepatosplenomegaly, enlarged thymus, and ascites; they were weak, strabismus-like, rounded, cachectic, lethargic, And he had trouble breathing.
[0221]
spleen. As shown in Table 2 below and in FIG. 10, abnormalities in the spleen were detected in all homozygous mutants, including increased body weight and increased spleen: body weight ratio compared to wild-type mice. These averaged 0.41% of wild-type control females against 0.61% of homozygous females and 0.52% of wild-type control females against homozygous mutant males. It was 0.26 percent of the male. The spleen weight and spleen: body weight ratio from day 68 to 189 were about 5-fold greater than in wild-type mice.
[0222]
Splenic lymphoid vesicles were expanded by an increased number of lymphocytes (hyperplasia) in the germinal center and at the marginal area of vesicles in homozygous mice.
[0223]
[Table 2]

liver. In the mutant mice, abnormalities in the liver were detected, including increased size, weight, and discoloration compared to wild-type mice. Liver weight and liver: body ratio were about 2-fold greater than in wild-type control mice (Table 2 and FIG. 11).
[0224]
kidney. Abnormalities in the kidney were detected in mutant mice, including increased size, weight, and discoloration compared to wild-type mice. The kidney: body weight ratio was about 1.5 times greater than in wild-type control mice (Table 2 and FIG. 12).
[0225]
Thymus. Abnormalities in the thymus were detected. Thymus weight and thymus: body weight ratio were about 10-30 fold greater than in wild type control mice (Table 1 and FIG. 13). There was moderate thymus atrophy with reduced cortical features. In addition, thymic cortical enlargement (hyperplasia) and associated medullary hypoplasia (atrophy) were observed in all homozygous mice. Thymic cortical lymphocytes were hyperplastic with increased mitotic activity and apoptosis.
[0226]
Bone and bone marrow. Abnormalities in bone and bone marrow were detected between days 68-189. The bone marrow was pale and the bone was brittle with white mass attached.
[0227]
body weight. The weight of male homozygous mice was approximately 60% of the weight of wild-type control mice. There was no difference in body weight between wild type control mice versus female homozygous mutant mice. Overall observations were incomplete for some mice.
[0228]
Serum chemistry. Clinically diseased homozygous mice had elevated levels of various analytes when compared to wild-type mice. A number of liver-related analytes were evaluated, including: alanine aminotransferase (ALP) in all four males and two of the three females; bilirubin in two males; and alkaline in one male Phosphatase (ALP). Aspartate aminotransferase, another liver-related analyte, was elevated in four males and two females. These various elevations are consistent with liver tumor cell infiltration. Cardiac tumor cell infiltration could also contribute to ALT elevation. Kidney-related analytes also rose. Three males had elevated blood urea nitrogen (BUN) and phosphate levels. This is probably related to tumor cell infiltration of the kidney and / or kidney.
[0229]
Expression. Total RNA was isolated from organs or tissues from adult C57B1 / 6 wild type mice. RNA was DNaseI treated and reverse transcribed using random primers. The resulting cDNA was examined for the absence of genomic contamination using primers specific for non-transcribed genomic mouse DNA. The cDNA was averaged for concentration using HPRT primers. RNA transcripts are found in the cerebellum, olfactory bulb, eye, heart, lung, liver, pancreas, kidney, thymus, lymph nodes, skin, gallbladder, bladder, pituitary, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, It was detectable in the cecum, testis, epididymis, seminal vesicles, coagulation gland, prostate, ovary, and uterus.
[0230]
Example 5 Generation and Analysis of Mice Containing BMP Gene Disruption
To investigate the role of BMP, disruptions in the BMP gene were created by homologous recombination. Specifically, transgenic mice containing a disruption in the BMP gene were generated. More specifically, as shown in FIGS. 15A-15B, a BMP-specific targeting construct capable of disrupting or altering a BMP gene (including SEQ ID NO: 17 in particular) was identified herein as An oligonucleotide sequence identified as SEQ ID NO: 19 or SEQ ID NO: 20 was generated in this construct using as a targeting arm (homologous sequence).
[0231]
This targeting construct was introduced into ES cells from the 129 / Sv- + P + Mgf-SLJ / J mouse subline to produce chimeric mice. F1 mice were generated by crossing with C57BL / 6 females, and F2 homozygous mutant mice were generated by crossing male and female F1 heterozygotes.
[0232]
Transgenic mice containing a disruption in the BMP gene were analyzed for phenotypic changes. The phenotype associated with the disruption in the nuclear receptor gene was determined. Homozygous mice exhibited at least one of the following phenotypes:
1) Unusual tail:
On day 49, 5 (2 females (10796, 10799) and 3 males (10829, 9) of the 9 homozygous mice examined when compared to age- and gender-matched wild-type control mice. 19478, 19479)) had an abnormal tail.
[0233]
On day 300 (actually 303-450 days of age), one male (10827) of the 9 homozygous mutant mice had an abnormal tail when compared to age- and gender-matched wild-type control mice. did.
[0234]
2) Weight:
Males and females at 49, 90, 180, and 300 days of age, homozygous mutants that tend to lose weight.
[0235]
Male and female homozygous mutants at 49, 90, 180, and 300 days of age tending to have a low weight / length ratio.
[0236]
3) Short length:
Male and female homozygous mutants at 49, 90, 180, and 300 days of age.
[0237]
More specifically, when compared to age (49, 90, 180, and 300 days of age) and gender-matched wild-type control mice, homozygous mutant mice at all time points had all three parameters (body weight) , Body length, and weight / body length ratio). In general, the differences between homozygous mutants and matched wild-type control mice were greater for males than for females. Body weight and weight / length ratio decreased for homozygous mutant males after 180 days of age. For each of the three parameters, the magnitude of the difference between the wild-type control male and the homozygous mutant male was greatest at 300 days of age.
[0238]
Example 6 Generation and Analysis of Mice Containing Airway Trypsin-Like Protease Gene Disruption
To investigate the role of airway trypsin-like protease, disruptions in the airway trypsin-like protease gene were created by homologous recombination. Specifically, transgenic mice containing a disruption in the airway trypsin-like protease gene were generated. More specifically, as shown in FIGS. 17A-17B, an airway trypsin-like protease-specific targeting construct that has the ability to disrupt or modify the airway trypsin-like protease gene (including SEQ ID NO: 21 in particular). The oligonucleotide sequence identified herein as SEQ ID NO: 22 or SEQ ID NO: 24 was generated using this targeting arm (homologous sequence) in this construct.
[0239]
This targeting construct was introduced into ES cells from the 129 / Sv- + P + Mgf-SLJ / J mouse subline to produce chimeric mice. F1 mice were generated by crossing with C57BL / 6 females, and F2 homozygous mutant mice were generated by crossing male and female F1 heterozygotes. Transgenic mice containing a disruption in the airway trypsin-like protease gene were analyzed for phenotypic changes.
[0240]
Example 7 Generation and Analysis of Mice Containing Aquaporin-8 Gene Disruption
To investigate the role of the aquaporin-8 gene, disruptions in the aquaporin-8 gene were created by homologous recombination. Specifically, transgenic mice containing a disruption in the aquaporin-8 gene were generated. More specifically, as shown in FIGS. 19A-B, an aquaporin-8 specific targeting construct capable of disrupting or altering the aquaporin-8 gene (specifically, including SEQ ID NO: 25) is described in the present invention. An oligonucleotide sequence identified herein as SEQ ID NO: 27 or SEQ ID NO: 28 was generated using this targeting arm (homologous sequence) in this construct.
[0241]
This targeting construct was introduced into ES cells from the 129 / Sv- + P + Mgf-SLJ / J mouse subline to produce chimeric mice. F1 mice were generated by crossing with C57BL / 6 females, and F2 homozygous mutant mice were generated by crossing male and female F1 heterozygotes.
[0242]
Transgenic mice containing a disruption in the aquaporin-8 gene were analyzed for phenotypic changes and expression patterns. The phenotype associated with the disruption in the nuclear receptor gene was determined. Homozygous mice exhibited at least one of the following phenotypes:
body weight. Homozygous mutant males tend to be heavier than wild-type control mice after 90 days of age.
[0243]
Expression. Total RNA was isolated from organs or tissues from adult C57B1 / 6 wild type mice. RNA was DNaseI treated and reverse transcribed using random primers. The resulting cDNA was examined for the absence of genomic contamination using primers specific for non-transcribed genomic mouse DNA. The cDNA was averaged for concentration using HPRT primers. RNA transcripts were detectable in eyes, liver, pancreas, thymus, gallbladder, stomach, large intestine, cecum, testis, seminal vesicle, ovary, and uterus.
[0244]
As will be apparent to those skilled in the art, various modifications of the above embodiments may be made without departing from the spirit and scope of the invention. These modifications and variations are within the scope of the present invention.
[Brief description of the drawings]
FIG.
FIG. 1 shows the polynucleotide sequence of the orphan anaphylatoxin C3a receptor (SEQ ID NO: 1). FIG. 1 also shows the amino acid sequence of the anaphylatoxin C3a receptor (SEQ ID NO: 2).
FIG. 2A
FIG. 2A shows the design of a targeting construct used to disrupt the anaphylatoxin C3a receptor gene.
FIG. 2B
FIG. 2B shows the design of a targeting construct used to disrupt the anaphylatoxin C3a receptor gene. FIG. 2B also shows the sequences identified as SEQ ID NO: 3 and SEQ ID NO: 4, which were used as targeting arms (homologous sequences) in the anaphylatoxin C3a receptor targeting construct.
FIG. 3
FIG. 3 shows a graph of thymus weight and body weight ratios for wild-type and heterozygous and homozygous mice with disruptions in the anaphylatoxin C3a receptor gene.
FIG. 4
FIG. 4 shows the polynucleotide sequence of the 5-HT5A gene (SEQ ID NO: 5). FIG. 4 also shows the amino acid sequence for the 5-HT5A gene (SEQ ID NO: 6).
FIG. 5A
FIG. 5A shows the design of a targeting construct used to disrupt the 5-HT5A gene.
FIG. 5B
FIG. 5B shows the design of a targeting construct used to disrupt the 5-HT5A gene. FIG. 5B also shows the sequences identified as SEQ ID NO: 7 and SEQ ID NO: 8, which were used as targeting arms (homologous sequences) in the 5-HT5A targeting construct.
FIG. 6
FIG. 6 shows the polynucleotide sequence of orphan cordin (SEQ ID NO: 9). FIG. 6 also shows the amino acid sequence of the cordin polypeptide (SEQ ID NO: 10).
FIG. 7A
FIG. 7A shows the design of a targeting construct used to disrupt the cordin gene.
FIG. 7B
FIG. 7B shows the design of the targeting construct used to disrupt the cordin gene. FIG. 7B also shows the sequences identified as SEQ ID NO: 11 and SEQ ID NO: 12, which were used as targeting arms (homologous sequences) in the cordin targeting construct.
FIG. 8
FIG. 8 shows the polynucleotide sequence of orphan RORγ (SEQ ID NO: 13). FIG. 8 also shows the amino acid sequence of RORγ (SEQ ID NO: 14).
FIG. 9A
FIG. 9A shows the design of a targeting construct used to disrupt the RORγ gene.
FIG. 9B
FIG. 9B shows the design of a targeting construct used to disrupt the RORγ gene. FIG. 9B also shows the sequences identified as SEQ ID NO: 15 and SEQ ID NO: 16, which were used as targeting arms (homologous sequences) in the RORγ targeting construct.
FIG. 10
FIG. 10 shows a graph of spleen weight and body weight ratios for wild-type and heterozygous and homozygous mice with disruptions in the RORγ gene.
FIG. 11
FIG. 11 shows graphs for liver weight and body weight ratios of wild-type mice and heterozygous and homozygous mice with disruption.
FIG.
FIG. 12 shows a graph for the ratio of kidney weight and body weight of wild-type and heterozygous and homozygous mice with a disruption in the RORγ gene.
FIG. 13
FIG. 13 shows a graph of thymus weight and body weight ratios for wild-type and heterozygous and homozygous mice with disruptions in the RORγ gene.
FIG. 14
FIG. 14 shows the polynucleotide sequence of the BMP gene (SEQ ID NO: 17). FIG. 14 also shows the amino acid sequence of the BMP polypeptide (SEQ ID NO: 18).
FIG. 15A
FIG. 15A shows the design of a targeting construct used to disrupt the BMP gene.
FIG. 15B
FIG. 15B shows the design of the targeting construct used to disrupt the BMP gene. FIG. 15B also shows the sequences identified as SEQ ID NO: 19 and SEQ ID NO: 20 that were used as targeting arms (homologous sequences) in the BMP targeting construct.
FIG.
FIG. 16 shows the polynucleotide sequence of the orphan airway trypsin-like protease (SEQ ID NO: 21). FIG. 16 also shows the amino acid sequence of this airway trypsin-like protease (SEQ ID NO: 22).
FIG. 17A
FIG. 17A shows the design of a targeting construct used to disrupt the airway trypsin-like protease gene.
FIG. 17B
FIG. 17B shows the design of a targeting construct used to disrupt the airway trypsin-like protease gene. FIG. 17B also shows the sequences identified as SEQ ID NO: 23 and SEQ ID NO: 24, which were used as targeting arms (homologous sequences) in the airway trypsin-like protease targeting construct.
FIG.
FIG. 18 shows the polynucleotide sequence of Aquaporin 8 (SEQ ID NO: 25). FIG. 18 also shows the amino acid sequence of aquaporin 8 (SEQ ID NO: 26).
FIG. 19A
FIG. 19A shows the design of a targeting construct used to disrupt the aquaporin 8 gene.
FIG. 19B
FIG. 19B shows the design of a targeting construct used to disrupt the aquaporin 8 gene. FIG. 19B also shows the sequences identified as SEQ ID NO: 27 and SEQ ID NO: 28 that were used as targeting arms (homologous sequences) in the Aquaporin 8 targeting construct.

Claims (91)

A targeting construct, comprising:
(A) a first polynucleotide sequence homologous to the target gene;
A targeting construct comprising: (b) a second polynucleotide sequence homologous to the target gene; and (c) a selectable marker. 2. The targeting construct of claim 1, wherein the targeting construct further comprises a screening marker. A method for producing a targeting construct for a target gene, the method comprising:
(A) obtaining a first polynucleotide sequence homologous to the target gene;
(B) obtaining a second polynucleotide sequence homologous to the target gene;
(C) providing a vector comprising a selectable marker; and (d) inserting the first sequence and the second sequence into the vector to produce the targeting construct. Method. A method for producing a targeting construct for a target gene, the method comprising:
(A) providing a polynucleotide comprising a first sequence homologous to a first region of the target gene and a second sequence homologous to a second region of the target gene; and (b) the first sequence. Inserting a positive selectable marker between and a second sequence to form a targeting construct. A cell that contains a disruption in the target gene. The cell according to claim 5, wherein the cell is a mouse cell. The cell according to claim 6, wherein the mouse cell is an embryonic stem cell. A non-human transgenic animal containing a disruption in the target gene. A cell derived from the non-human transgenic animal according to claim 8. A method for producing a transgenic mouse comprising a disruption in a target gene, comprising:
(A) introducing the targeting construct of claim 1 into a cell;
(B) introducing the cells into blastocysts;
(C) transplanting the obtained blastocyst into a pseudopregnant mouse, wherein the pseudopregnant mouse produces a chimeric mouse; and (d) breeding the chimeric mouse to obtain a transgenic mouse A method comprising producing A method for identifying an agent that modulates expression of a target gene, the method comprising:
(A) providing a non-human transgenic animal containing a disruption in the target gene;
(B) administering an agent to the non-human transgenic animal; and (c) determining whether expression of the disrupted target gene in the non-human transgenic animal is modulated. . A method for identifying an agent that modulates the function of a target gene, the method comprising:
(A) providing a non-human transgenic animal containing a disruption in the target gene;
(B) administering an agent to the non-human transgenic animal; and (c) determining whether the function of the disrupted target gene in the non-human transgenic animal is modulated. . A method for identifying an agent that modulates expression of a target gene, the method comprising:
(A) providing a cell containing a disruption in the target gene;
(B) contacting the cell with an agent; and (c) determining whether the expression of the target gene is modulated. A method for identifying an agent that modulates the function of a target gene, the method comprising:
(A) providing a cell containing a disruption in the target gene;
(B) contacting the cell with an agent; and (c) determining whether the function of the target gene is modulated. 15. The method of claim 13 or claim 14, wherein the cells are from the non-human transgenic animal of claim 8. An agent identified by the method of claim 11, claim 12, claim 13 or claim 14. An agent that regulates the activity of a target gene. A transgenic mouse comprising a disruption in the anaphylatoxin C3a receptor gene, wherein the transgenic mouse exhibits thymic abnormalities. 19. The transgenic mouse of claim 18, wherein said thymic abnormality is a decrease in thymus weight as compared to a wild-type mouse. 19. The transgenic mouse of claim 18, wherein said thymic abnormality is a decrease in thymus to body weight ratio compared to a wild-type mouse. A transgenic mouse comprising a disruption in the anaphylatoxin C3a receptor gene, wherein the transgenic mouse exhibits increased susceptibility to seizures. 22. The transgenic mouse of claim 21, wherein said mouse shows a seizure-like response at a lower dose of metrazole as compared to a wild-type mouse. A transgenic mouse comprising a disruption in the anaphylatoxin C3a receptor gene, wherein the transgenic mouse exhibits a stimulatory processing defect as compared to a wild-type mouse. 24. The transgenic mouse of claim 23, wherein said stimulus processing defect is similar to the stimulus processing defect observed in schizophrenia. 24. The transgenic mouse of claim 23, wherein said mouse exhibits reduced prepulse inhibition as compared to a wild-type mouse. A method for producing a transgenic mouse comprising a disruption in the anaphylatoxin C3a receptor gene, wherein said transgenic mouse has at least the following phenotype: thymic abnormalities, increased susceptibility to seizures, or stimulatory processing defects. Show one, where the method comprises:
(A) introducing an anaphylatoxin C3a receptor gene targeting construct into a cell;
(B) introducing the cells into blastocysts;
(C) transplanting the obtained blastocyst into a pseudopregnant mouse, wherein the pseudopregnant mouse produces a chimeric mouse; and (d) breeding the chimeric mouse to anaphylaxis. Producing a transgenic mouse containing a disruption in the toxin C3a receptor gene. A cell derived from the transgenic mouse according to claim 18 or 26. A method for identifying an agent that ameliorates a phenotype associated with a disruption in an anaphylatoxin C3a receptor gene, the method comprising:
(A) administering the drug to a transgenic mouse that contains a disruption in the anaphylatoxin C3a receptor gene; and (b) the drug has the following phenotype: thymic abnormalities, increased susceptibility to seizures, or lack of stimulus processing. Deciding whether to improve at least one. A method for identifying an agent that modulates anaphylatoxin C3a receptor expression, comprising:
(A) administering the agent to a transgenic mouse that contains a disruption in the anaphylatoxin C3a receptor gene; and (b) determining whether the agent modulates anaphylatoxin C3a receptor expression in the transgenic mouse. Wherein the agent has an effect on at least one of the following behaviors: susceptibility to stroke and stimulus processing. A method for identifying an agent that modulates a behavior associated with disruption in an anaphylatoxin C3a receptor gene, the method comprising:
(A) administering the agent to a transgenic mouse that contains a disruption in the anaphylatoxin C3a receptor gene; and (b) whether the agent modulates at least one of the following behaviors: susceptibility to stroke and stimulus processing. Steps to determine:
A method comprising: A method for identifying an agent that modulates anaphylatoxin C3a receptor gene function, comprising:
(A) providing a cell containing a disruption in the anaphylatoxin C3a receptor gene;
(B) contacting the cells with an agent; and (c) determining whether the agent modulates anaphylatoxin C3a receptor gene function, wherein the agent comprises an anaphylatoxin C3a receptor gene. Modulating the phenotype associated with disruption in the method. 32. The method of claim 31, wherein the phenotype comprises at least one of thymic abnormalities, increased susceptibility to seizures, and stimulatory processing deficits. An agent identified by the method according to claim 28, 29, 30 or 31. An agent that modulates the activity of an anaphylatoxin C3a receptor gene. A method for ameliorating a disease associated with a mutation in an anaphylatoxin C3a receptor gene, comprising administering to a subject in need thereof a therapeutically effective amount of an agent that modulates the activity of an anaphylatoxin C3a receptor gene. A method comprising: A transgenic mouse comprising a disruption in the cordin gene, wherein the transgenic mouse exhibits at least one of the following phenotypes: nociceptive disorder and anxiety disorder. A method for producing a transgenic mouse comprising a disruption in the cordin gene, wherein said transgenic mouse exhibits at least one of the following phenotypes: nociceptive disorder and anxiety disorder, comprising:
(A) introducing the cordin gene targeting construct into cells;
(B) introducing the cells into blastocysts;
(C) transplanting the obtained blastocyst into a pseudopregnant mouse, wherein the pseudopregnant mouse lays a chimeric mouse; and (d) breeding the chimeric mouse, Producing the transgenic mouse containing a disruption in the gene. A cell derived from the transgenic mouse according to claim 36 or 37. A method for identifying an agent that ameliorates a phenotype associated with a disruption in the cordin gene, comprising:
(A) administering the agent to a transgenic mouse containing a disruption in the cordin gene; and (b) determining whether the agent improves at least one of the following phenotypes: nociceptive disorder and anxiety disorder A method comprising the step of determining. A method for identifying an agent that modulates cordin expression, comprising:
(A) administering the agent to a transgenic mouse containing a disruption in the cordin gene; and (b) determining whether the agent modulates cordin expression in the transgenic mouse, comprising: Wherein the agent has an effect on at least one of the following behaviors: response to pain and anxiety. A method for identifying an agent that modulates a behavior associated with a disruption in the cordin gene, comprising:
(A) administering the drug to a transgenic mouse that contains a disruption in the cordin gene; and (b) determining whether the drug modulates coordination and equilibrium in the transgenic mouse. ,Method. A method for identifying an agent that modulates cordin gene function, comprising:
(A) providing a cell containing a disruption in the cordin gene;
(B) contacting the cells with an agent; and (c) determining whether the agent modulates cordin gene function, wherein the agent is associated with a disruption in the cordin gene. Modulating the phenotype of the cells. 43. The method of claim 42, wherein said phenotype comprises at least one of a nociceptive disorder and an anxiety disorder. An agent identified by the method of claim 39, 40, 41, or 42. A transgenic mouse comprising a disruption in the cordin gene, wherein said transgenic mouse exhibits a reduced response to pain. A drug that regulates the activity of the cordin gene. A method of ameliorating a disease associated with a mutation in the cordin gene, the method comprising administering to a subject in need thereof a therapeutically effective amount of an agent that modulates the activity of the cordin gene. A transgenic mouse containing a disruption in the RORγ gene, wherein the transgenic mouse has the following phenotype: spleen abnormality, kidney abnormality, spleen abnormality, liver abnormality, thymus abnormality, lymph node abnormality, lymphocyte abnormality. A transgenic mouse showing at least one of an abnormality in bone marrow or an abnormality in bone. 49. The transgenic mouse of claim 48, wherein said spleen abnormality is an increase in spleen weight as compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein said spleen abnormality is an increase in spleen size as compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein the spleen abnormality is an increase in spleen to body weight ratio compared to a wild type mouse. 49. The transgenic mouse of claim 48, wherein said kidney abnormality is an increase in kidney weight compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein said kidney abnormality is an increase in kidney size as compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein said renal abnormality is an increase in kidney to body weight ratio compared to a wild type mouse. 49. The transgenic mouse of claim 48, wherein said liver abnormality is an increase in liver weight as compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein said liver abnormality is an increase in liver size as compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein said liver abnormality is an increase in liver-to-body weight ratio compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein said thymic abnormality is an increase in thymus weight as compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein said thymic abnormality is an increase in thymus size as compared to a wild-type mouse. 49. The transgenic mouse of claim 48, wherein said thymic abnormality is an increase in thymus to body weight ratio compared to a wild type mouse. 49. The transgenic mouse of claim 48, wherein the thymic abnormalities are thymic cortical enlargement and medullary decrease as compared to wild-type mice. 49. The transgenic mouse of claim 48, wherein said lymph node abnormality is lymph node deficiency as compared to a wild type mouse. 49. The transgenic mouse of claim 48, wherein said lymph node abnormality is the absence of a lymph node. Said lymph node abnormality is a deficiency of gastrointestinal tract-associated lymphoid tissue compared to wild type mice. A transgenic mouse according to claim 48. 49. The transgenic mouse of claim 48, wherein said lymphocyte abnormality comprises lymph infiltration. 49. The transgenic mouse of claim 48, wherein said lymphocyte abnormality is consistent with lymphoma. Symptoms of the following diseases: at least one of unhealthy dressing, impaired function, increased chest girth and increased abdomen circumference, hepatosplenomegaly, enlarged thymus, ascites, weakness, strabismus, stoop, cachexia, lethargy, and breathing disorders 67. The transgenic mouse of claim 66, further comprising: 49. The transgenic mouse of claim 48, wherein said bone marrow is pale. 49. The transgenic mouse of claim 48, wherein said bone abnormality is brittle. 49. The transgenic mouse of claim 48, wherein the bone abnormality is associated with a white mass. A method for producing a transgenic mouse containing a disruption in the RORγ gene, wherein the transgenic mouse has the following phenotypes: spleen abnormality, kidney abnormality, spleen abnormality, liver abnormality, thymus abnormality, lymph node At least one of the following: abnormalities in lymphocytes, abnormalities in lymphocytes, abnormalities in bone marrow, or abnormalities in bones.
(A) introducing a RORγ gene targeting construct into a cell;
(B) introducing the cells into blastocysts;
(C) transplanting the obtained blastocyst into a pseudopregnant mouse, wherein the pseudopregnant mouse gives birth to a chimeric mouse; and (d) breeding the chimeric mouse to obtain a RORγ gene. Producing a transgenic mouse containing a disruption therein. A cell derived from the transgenic mouse according to claim 48 or claim 71. A method for identifying an agent that ameliorates a phenotype associated with a disruption in the RORγ gene, comprising:
(A) administering the drug to a transgenic mouse having a disruption in the RORγ gene; and (b) the drug having the following phenotype: spleen abnormality, kidney abnormality, spleen abnormality, liver abnormality, thymic abnormality, Deciding whether to ameliorate at least one of an abnormality in a lymph node, an abnormality in lymphocytes, an abnormality in bone marrow, or an abnormality in bone. A method for identifying an agent that ameliorates a phenotype associated with a disruption in the RORγ gene, comprising:
(A) administering the drug to a transgenic mouse containing a disruption in the RORγ gene; and (b) the drug having the following phenotype: increased serum alanine aminotransferase, increased serum alkaline phosphatase, increased serum. Deciding whether to improve at least one of aspartate transferase, elevated blood urea nitrogen and elevated blood phosphorus. A method for identifying an agent that ameliorates a phenotype associated with a disruption in the RORγ gene, comprising:
(A) administering a drug to a transgenic mouse containing a disruption in the RORγ gene; and (b) determining whether the drug regulates RORγ expression in the transgenic mouse. Where the drug is used for the symptoms of the following diseases: unhealthy dressing, reduced function, increased chest girth and increased abdomen, hepatosplenomegaly, enlarged thymus, ascites, weakness, strabismus, stoop, cachexia, lethargy And having an effect on at least one of respiratory disorders. A method for identifying an agent that modulates RORγ gene function, comprising:
(A) providing a cell containing a disruption in the RORγ gene;
(B) contacting the cells with an agent; and (c) determining whether the agent modulates RORγ gene function, wherein the agent is associated with a disruption in the RORγ gene. How to control the phenotype you do. 46. The method of claim 45, wherein the phenotype comprises spleen abnormalities, kidney abnormalities, spleen abnormalities, liver abnormalities, thymic abnormalities, abnormalities in lymph nodes, abnormalities in lymphocytes, abnormalities in bone marrow or bones. 46. The method of claim 45, wherein said phenotype comprises a lymphoma. An agent identified by the method of claim 42, 43, 44, or 45. A transgenic mouse comprising a disruption in the RORγ gene, wherein said transgenic mouse exhibits lymphoma. An agent that regulates the activity of the RORγ gene. A method for ameliorating a disease associated with a mutation in the RORγ gene, comprising administering to a subject in need thereof a therapeutically effective amount of an agent that modulates the activity of the RORγ gene. Method. A transgenic mouse comprising a disruption in the BMP gene, wherein the transgenic mouse exhibits at least one of the following phenotypes: abnormal tail, low weight or short body length. A method of producing a transgenic mouse comprising a disruption in the BMP gene, wherein said transgenic mouse exhibits at least one of the following phenotypes: abnormal tail, low body weight or short body length, Less than:
(A) introducing a BMP gene targeting construct into a cell;
(B) introducing the cells into blastocysts;
(C) transplanting the obtained blastocyst into a pseudopregnant mouse, wherein the pseudopregnant mouse produces a chimeric mouse; and (d) breeding the chimeric mouse and disrupting it into the BMP gene Producing a transgenic mouse comprising: A cell derived from the transgenic mouse according to claim 83 or 84. A method of identifying an agent that ameliorates a phenotype associated with a disruption in a BMP gene, comprising:
(A) administering the agent to a transgenic mouse comprising a disruption in the BMP gene; and (b) determining whether the agent improves at least one of the following phenotypes: abnormal tail, low body weight or short body length Deciding whether or not they are not. A method for identifying an agent that modulates BMP gene function, comprising:
(A) providing a cell containing a disruption in the BMP gene;
(B) contacting the cells with a drug; and (c) determining whether the drug modulates BMP gene function, wherein the drug is associated with a disruption in the BMP gene. Modulating the phenotype of the cells. 32. The method of claim 31, wherein the phenotype comprises at least one of an abnormal tail, low weight, or short body length. 88. An agent identified by the method of claim 86 or claim 87. An agent that regulates the activity of a BMP gene. A method of ameliorating a disease associated with a mutation in a BMP gene, the method comprising administering to a subject in need thereof a therapeutically effective amount of an agent that modulates the activity of the BMP gene. .

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