JP2006121964A - Anti-obesity transgenic pig - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a transgenic pig which can be used as an anti-obesity or anti-diabetic animal model and a method for producing the same.
A transgenic pig is one into which a leptin gene or an adiponectin gene has been introduced. Further, a method for producing a transgenic pig was obtained by introducing a vector in which the leptin gene or adiponectin gene was inserted downstream of a promoter functioning in pig cells into an egg by a sperm vector method or a pronuclear injection method. Generating an individual from a fertilized egg.
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Description

  The present invention relates to an anti-obesity or anti-diabetic transgenic pig.

  In transgenic mice, overexpression of adipocytokines secreted from adipose tissue such as leptin and adiponectin has been observed to have anti-obesity, insulin sensitivity-enhancement and anti-arteriosclerosis effects. Collecting.

Pro. Natl. Acad. Sci. USA Vol. 95, 11852-11857, 1998, ELLA IOFFE et al. Abnormal regulation of the leptin gene in the pathogenesis of obesity. J. Bio. Chem. Vol 278, 2461-2468, 2003, Toshimasa Yamauchi et al. Globular Adiponectin Protected ob / ob Mice from Diabetes and ApoE-deficient Mice from Atherosclerosis.

  Transgenic mice into which the leptin gene or adiponectin gene has been introduced have been produced, but the differences between rodent mice and humans are large, and there are many unreasonable parts as human models. Pigs, on the other hand, are considered to be genetically and physiologically closer to humans, and are omnivorous in eating habits and eat the same foods as humans. It is considered to be a good model for verifying a simple diet method. In addition, since it responds sensitively to stress, it is considered to be a good model animal to investigate how the influence of stress in modern society with stress is related to obesity and diabetes. However, no anti-obesity or anti-diabetic transgenic pigs have been obtained. Accordingly, an object of the present invention is to provide a transgenic pig that can be used as an anti-obesity or anti-diabetic animal model.

  The inventors of the present application introduce a vector in which the leptin gene or adiponectin gene is inserted downstream of a promoter that functions in swine cells into a fertilized egg by a sperm vector method or a pronuclear injection method, and an individual is transferred from the fertilized egg. Thus, the present inventors have successfully produced a transgenic pig introduced with a leptin gene or adiponectin gene, and completed the present invention.

  That is, the present invention provides a transgenic pig introduced with a leptin gene or adiponectin gene. In addition, the present invention introduces a vector in which the leptin gene or adiponectin gene is inserted downstream of a promoter that functions in swine cells into an egg by a sperm vector method or a pronuclear injection method, and an individual is obtained from the resulting fertilized egg. A method for producing a transgenic pig is provided.

  According to the present invention, a transgenic pig introduced with a leptin gene or an adiponectin gene was provided for the first time. Since pigs are genetically and physiologically close to humans, the transgenic pigs of the present invention elucidate the effects of adipocytokines, such as leptin and adiponectin, which are mainly involved in obesity mechanisms in humans, Therefore, the present invention is considered to contribute greatly to human obesity and diabetes research.

  As described above, the transgenic pig of the present invention has an exogenous leptin gene or adiponectin gene introduced therein. In the present specification, the “gene” includes not only genomic genes but also cDNAs. Rather, it is convenient and preferable to use cDNAs. Moreover, the gene is preferably a porcine-derived gene. The porcine adiponectin gene itself is known and its nucleotide sequence is also known (NCBI Accession No. AY135647) (SEQ ID NO: 1). The porcine leptin gene itself is also known and its nucleotide sequence is also known (NCBI Accession No. AF026976) (SEQ ID NO: 3). In the present invention, genes having these nucleotide sequences can be used as they are.

  In general, in the amino acid sequence of a protein having physiological activity, even if a small number of amino acids are substituted or deleted, or even if a small number of amino acids are inserted or added, the physiological activity is maintained. It is well known to those skilled in the art. Accordingly, in a known amino acid sequence of leptin (SEQ ID NO: 2), a polypeptide having an amino acid sequence in which a small number of amino acids are substituted or deleted, or a small number of amino acids are inserted or added, and the leptin activity A gene encoding a polypeptide having the above can also be used in the present invention, and such a gene is also included in the “leptin gene” referred to in the present specification. In this case, in the amino acid sequence of SEQ ID NO: 2, a small number of amino acids are substituted or deleted, or a small number of amino acids are inserted or added, and the amino acid sequence of SEQ ID NO: 2 is 90% or more. More preferably, it is 95% or more. Amino acid sequence identity can be easily calculated using well-known computer software such as BLAST, and such software is also available on the Internet. Further, when a small number of amino acids are substituted, deleted and / or inserted, the total number of amino acids to be substituted, deleted and / or inserted is preferably 1 to several. In addition, 20 kinds of amino acids constituting natural proteins are neutral amino acids having low polarity side chains (Gly, Ile, Val, Leu, ala, Met, Pro), neutral amino acids having hydrophilic side chains (Asn , Gln, Thr, Ser, Tyr Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp) In many cases, the physiological activity of the protein is not substantially changed if the substitution is performed within each group. The same applies to the adiponectin gene, and a polypeptide having an amino acid sequence in which a small number of amino acids are substituted or deleted, or a small number of amino acids are inserted or added in the known amino acid sequence of adiponectin (SEQ ID NO: 4) Thus, a gene encoding a polypeptide having adiponectin activity can also be used in the present invention, and such a gene is also included in the “adiponectin gene” as used herein. In this case, in the amino acid sequence of SEQ ID NO: 4, a small number of amino acids are substituted or deleted, or a small number of amino acids are inserted or added, and the amino acid sequence of SEQ ID NO: 4 is 90% or more. More preferably, it is 95% or more. Further, when a small number of amino acids are substituted, deleted and / or inserted, the total number of amino acids to be substituted, deleted and / or inserted is preferably 1 to several.

  In addition, when the leptin gene or adiponectin gene is in the form of a fusion gene linked to a reporter gene, fertilized eggs into which the leptin gene or adiponectin gene has been introduced can be easily identified using the product of such a reporter gene. So convenient. As the reporter gene, various known reporter genes can be used. Preferably, a GFP (green fluorescent protein) gene or an EGFP (enhanced green fluorescent protein) gene whose mutant is increased in fluorescence can be used. . Since expression vectors incorporating these reporter genes are commercially available, they can be used.

  The transgenic pig of the present invention was obtained by introducing a vector in which the leptin gene or adiponectin gene was inserted downstream of a promoter that functions in pig cells into the sperm by co-culturing with pig sperm. It can be produced by fertilizing pig sperm into a pig egg, culturing the resulting fertilized egg into an embryo, introducing this embryo into the uterus of a pig, and pregnancy and delivery (sperm vector method). Since expression vectors having a reporter gene introduced downstream of a promoter that functions in pig cells are commercially available, by inserting a leptin gene or adiponectin gene into the cloning site of such a commercially available vector, sperm An expression vector used in the vector method can be obtained. Details of the sperm vector method used for the production of transgenic pigs are described in the examples below. Alternatively, a fertilized egg into which a leptin gene or adiponectin gene has been introduced can also be obtained by microinjecting the expression vector into which the leptin gene or adiponectin gene has been introduced into the pronucleus of a pig fertilized egg (pronuclear injection method). ) The transgenic pig of the present invention can also be obtained by generating an individual from this fertilized egg. Details of this method are also described in the examples below.

  Since the leptin gene or adiponectin gene has been introduced into the transgenic pig of the present invention, leptin or adiponectin is overproduced, resulting in anti-obesity or anti-diabetic properties.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Production of transgenic pigs with adiponectin gene transfer by sperm vector method. Improved NCSU23 culture solution (Kurome M, Fujimura T, Murakami H, Takahagi Y, Wako N, Ochiai T, Miyazaki K, Nagashima H. Comparison of electro-fusion and intracytoplasmic nuclear Cloning and Stem Cells 2003; 5: 367-378. Composition is described later) or TCM199 medium (Kurihara T, Kurome M, Wako N, Ochiai T, Mizuno K, Fujimura T, Takahagi Y, Murakami H, Kano K, Miyagawa S, Shirakura R, Nagashima H. Developmental competence of in vitro matured porcine oocytes after electrical activation. J Reprod Dev 2002; 48: 271-279, composition is described later) Activation by DC pulse (150 V / mm, 100 μsec) followed by treatment with 7.5 μg / ml cytochalasin B for 3-4 hours. Activated oocytes were cultured for 7 days. Part of this was introduced into the oviduct of a young sow for fertilization in order to examine the cell's ability to grow in vitro and in vivo. BTS (Pursel VG, Johnson LA. Freezing of boar spermatozoa: Freezing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 1975; 40: 99-102, composition is described later) As mentioned above, composition is described later) Pig spermatozoa frozen in solution is adjusted to a concentration of 2-5 x 10 ^{5 and} co-cultured for 5 minutes with adiponectin-GFP DNA (2.5 ng / μl) (production method described later) did. After that, the isolated sperm was injected into the IVM oocyte with a piezo-type fine cell manipulator (micromanipulator) and activated by electrical stimulation in the same manner as described above. Oocytes injected with sperm were cultured in NCSN23 medium for 6 days and developed into blastocysts. The expression of the adiponectin gene was confirmed by fluorescence microscopy with the expression of GFP. In addition, messenger RNA in blastocysts was extracted and the presence of the adiponectin gene was confirmed by PCR.

  Nine recipient pigs were transplanted with 170 parthenogenetic eggs and laparotomy was performed 16-18 days later. As a result, 4 to 40 (23.5%) somite stage fetuses were obtained. When 70 activated oocytes were cultured for 7 days, 30 of them (42.9%) developed into blastocysts.

  From oocytes treated with 160 adiponectin gene-injected spermatozoa (under the conditions described above), 30 (18.8%) blastocysts were taken, of which 15 (50%) had GFP fluorescence. Of the blastocysts with fluorescence, the presence of adiponectin DNA was confirmed by PCR, and 10 (67%) were found to exist. As a control experiment, blastocysts having no GFP fluorescence appeared from oocytes injected with sperm that did not co-exist with adiponectin-GFP DNA.

  As a result of transplanting 250 adiponectin gene-introduced embryos to 4 recipients, 4 offspring were obtained, 2 of which were transgenic individuals.

The adiponectin expression vector used in the sperm vector method described above was prepared as follows. Porcine adipocyte-derived mRNA was extracted and purified by ISOGEN (Nippon Gene) according to the protocol. Furthermore, first strand cDNA was synthesized from the obtained mRNA by SuperScriptII RNaseH ^- reverse transcriptase (Invitrogen). The porcine adiponectin (NCBI Accession No. AY135647) cDNA was cloned by PCR using PfuTurbo DNA Polymerase (Toyobo Co., Ltd.) using the previously prepared porcine adipocyte-derived first strand cDNA pig as a template. At the time of cloning, a BamHI tag was attached to the five ends of the sense primer and the antisense primer so that they could be inserted into an EGFP expression vector: pEGFP-N1 (Nippon Becton Dickinson). The two primers are as follows (sense primer: ggatccaggggctcaggatgctgttg, antisense primer: tggatcctcaatgttgtggtagagaag). The obtained PCR product was subcloned into pCR4Blunt-TOPO (Invitrogen Corp.), and the sequence was confirmed by sequencing. The clone whose sequence was confirmed was excised with BamHI and ligated to pEGF-N1 treated with BamHI / CIAP (Calf Intestine Alkaline Phosphatase) to complete a fusion protein expression vector of porcine adiponectin and EGFP.

NCSU23 culture composition
NaCl 108.73 mM, KCl 4.78 mM, CaCl ₂・ 2H ₂ O 1.70 mM, MgSO ₄・ 7H ₂ O 1.19 mM, NaHCO ₃ 25.07 mM, KH ₂ PO ₄ 1.19 mM, glucose 5.55 mM, glutamine 1.00 mM, taurine 7.00 mM, Hipotaurine 5.00 mM, BSA 0.4%, penicillin G 100 IU / L, streptomycin 50 mg / L

TCM199 culture composition
CaCl ₂ (anhydrous) 200.00 mg / L, Fe ( NO 3) 3 · 9H 2 O 0.72 mg / L, KCl 400.00 mg / L, MgSO 4 ( anhydrous) 97.67 mg / L, NaCl 6800.00 mg / L, NaH 2 PO ₄ -H ₂ O 140.00 mg / L, adenosine sulfate 10.00 mg / L, ATP (2 Na salt) 1.00 mg / L, adenylate 0.20 mg / L, cholesterol 0.20 mg / L, deoxyribose 0.50 mg / L, D- Glucose 1000.00 mg / L, Glutathione (GSH) 0.05 mg / L, Guanine / HCl 0.30 mg / L, Hypoxanthine (Na salt) 0.354 mg / L, Phenol red 20.00 mg / L, Ribose 0.50 mg / L, Sodium acetate 50.00 mg / L, thymine 0.30 mg / L, Tween 80 (registered trademark) 20.00 mg / L, uracil 0.30 mg / L, xanthine (Na salt) 0.344 mg / L, DL-alanine 50.00 mg / L, L-arginine / HCl 70.00 mg / L, DL-aspartic acid 60.00 mg / L, L-cysteine / HCl / H ₂ O 0.11 mg / L, L-cystine / 2HCl 26.00 mg / L, DL-glutamic acid / H ₂ O 150.00 mg / L, L-glutamine 100.00 mg / L, glycine 50.00 mg / L, L-histidine _{HCl · H 2 O 21.88 mg /} L, L- hydroxyproline 10.00 mg / L, DL- isoleucine 40.00 mg / L, DL- leucine 120.00 mg / L, L- lysine · HCl 70.00 mg / L, DL- methionine 30.00 mg / L, DL-phenylalanine 50.00 mg / L, L-proline 40.00 mg / L, DL-serine 50.00 mg / L, DL-threonine 60.00 mg / L, DL-tryptophan 20.00 mg / L, L-tyrosine (2Na salt) 57.88 mg / L, DL-valine 50.00 mg / L, ascorbic acid 0.05 mg / L, α-tocopherol phosphate (2Na salt) 0.01 mg / L, d-biotin 0.01 mg / L, calciferol 0.10 mg / L, p- Calcium pantothenate 0.01 mg / L, choline chloride 0.50 mg / L, folic acid 0.01 mg / L, i-inositol 0.05 mg / L, menadione 0.01 mg / L, niacin 0.025 mg / L, niacinamide 0.025 mg / L, p- Aminobenzoic acid 0.05 mg / L, pyridoxal / HCl 0.025 mg / L, pyridoxine / HCl 0.025 mg / L, riboflavin 0.01 mg / L, thiamine / HCl 0.01 mg / L, vitamin A (acetate) 0.14 mg / L

BTS solution composition anhydrous dextrose 3.7 g / 100 mL, sodium citrate dihydrate 0.6 g / 100 mL, sodium bicarbonate 0.125 g / 100 mL, EDTA 2Na 0.125 g / 100 mL, potassium chloride 0.075 g / 100 mL

BF5 solution composition
Ter-N-tris (hydroxymethyl) methyl 2-aminoethane sulfonic acid 1.2 g / 100 mL, tris (hydroxymethyl) aminomethane 0.2 g / 100 mL, anhydrous dextrose 3.2 g / 100 mL, egg yolk 20 mL / 100 mL, Orbus BS Paste 0.5 mL / 100 mL

Production of leptin transgenic transgenic pigs by pronuclear injection method To produce leptin transgenic transgenic pigs, pronuclear injection method (Murakami H, Nagashima H, Takahagi Y, Miyagawa S, Fujimura T, Toyomura K, Nakai R , Yamada M, Kurihara T, Shigehisa T, Okabe M, Seya T, Shirakura R, Kinoshita T. Transgenic pigs expressing human decay-accelerating factor regulated by porcine MCP gene promoter. Mol Reprod Dev 2002; 61: 302-311) This was performed as follows. That is, mature female pigs (Landrace / Large White x Duroc species) in estrus were mated with males, and fertilized eggs were taken out from female oviducts the next day and treated with hyaluronidase (300 units / ml) to remove cumulus cells. Among these eggs, those with a clear pronucleus were used for the experiment. The fertilized egg thus obtained was aspirated and fixed to a round pipette (diameter 150 μm) using a micromanipulator. Insert the tip of the injection needle through the zona pellucida and yolk and insert it into the pronucleus of the egg, leptin-GFP DNA (diluted with Tris-EDTA buffer pH 7.4 to 5-10 ng / μL) (production method) Was injected). The fertilized eggs after gene injection were cultured in NCSU23 medium for 1-2 days and returned to the oviduct of estrus synchronized sows.

  In order to see the effectiveness of microinjection into the pronucleus, eggs injected with DNA were cultured for 16-24 hours, and the expression of GFP was observed and confirmed with a fluorescence microscope. Of the total of 512 eggs into which genes were introduced by microinjection, 298 (58.2%) showed GFP fluorescence.

  As a result of transplanting these 298 microinjected eggs into 4 recipient sows, 2 became pregnant and a total of 8 pups were obtained. As a result of the analysis, one was a transgenic pig.

  The leptin gene expression vector used for the pronuclear injection method was prepared as follows. The porcine leptin (NCBI Accession No. AF026976) cDNA was cloned by PCR using PfuTurbo DNA Polymerase (Toyobo Co., Ltd.) using the previously prepared porcine adipocyte-derived first strand cDNA as a template. At the time of cloning, a BamHI tag was attached to the five ends of the sense primer and the antisense primer so that they could be inserted into an EGFP expression vector: pEGFP-N1 (Nippon Becton Dickinson). The two primers are as follows (sense primer: ggatccaaaggaaaatgcgctgtgga, antisense primer: tggatcccagccagggctgaggtccag). The obtained PCR product was subcloned into pCR4Blunt-TOPO (manufactured by Invitrogen), and the sequence was confirmed by sequencing. The clone whose sequence was confirmed was excised with BamHI, and ligated to pEGF-N1 treated with BamHI / CIAP (Calf Intestine Alkaline Phosphatase) to complete a fusion protein expression vector of porcine leptin and EGFP.

Claims (6)

  A transgenic pig introduced with a leptin gene or an adiponectin gene.   The transgenic pig according to claim 1, wherein the leptin gene or adiponectin gene is cDNA.   The transgenic pig according to claim 1 or 2, wherein the leptin gene or adiponectin gene is a gene derived from pig.   The transgenic pig according to any one of claims 1 to 3, wherein a reporter gene is linked to the leptin gene or the adiponectin gene.   A vector in which the leptin gene or adiponectin gene is inserted downstream of a promoter that functions in swine cells is introduced into an egg by the sperm vector method or the pronuclear injection method, and an individual is generated from the resulting fertilized egg. The transgenic pig according to any one of claims 1 to 4. Including introducing a vector in which the leptin gene or adiponectin gene is inserted downstream of a promoter that functions in pig cells into an egg by a sperm vector method or a pronuclear injection method, and generating an individual from the resulting fertilized egg A method for producing a transgenic pig.