[go: up one dir, main page]

WO1996017946A1 - Produit de construction de transgenes et son expression amelioree - Google Patents

Produit de construction de transgenes et son expression amelioree Download PDF

Info

Publication number
WO1996017946A1
WO1996017946A1 PCT/GB1995/002839 GB9502839W WO9617946A1 WO 1996017946 A1 WO1996017946 A1 WO 1996017946A1 GB 9502839 W GB9502839 W GB 9502839W WO 9617946 A1 WO9617946 A1 WO 9617946A1
Authority
WO
WIPO (PCT)
Prior art keywords
region
protein encoding
expression
transgene
encoding region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB1995/002839
Other languages
English (en)
Inventor
Alan Colman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PPL Therapeutics Scotland Ltd
Original Assignee
PPL Therapeutics Scotland Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by PPL Therapeutics Scotland Ltd filed Critical PPL Therapeutics Scotland Ltd
Priority to AU39906/95A priority Critical patent/AU3990695A/en
Publication of WO1996017946A1 publication Critical patent/WO1996017946A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/206—Pseudochromosomes, minichrosomosomes of yeast origin, e.g. YAC, 2u

Definitions

  • the present invention relates to the expression of exogenous DNA (a transgene) in a host cell, and in particular relates to the expression of exogenous DNA in a transgenic animal.
  • Immunosuppressive drugs are used to reduce the occurrence and severity of the long term rejection process and use of such drugs has a number of drawbacks.
  • the immunosuppressive drugs act by decreasing the activity of the host's immune system generally, leaving the host more vulnerable to infections and the continued use of such drugs is essential throughout the whole life of the host and this can be restrictive. Even where immunosuppressive drugs are used, their use does not guarantee that the transplanted organ will not be rejected.
  • the donor's organs are characterised by particular antigenic epitopes - if these epitopes are recognised by the immune system of the host as being of foreign or "non-self" origin, activation of the host's immune system will lead to a reaction against the donated organ, possibly causing the destruction and/or rejection of that organ.
  • organs suitable for transplantation into humans are sourced primarily from other human bodies because of the obvious physiological and biochemical match and since the epitopes of non-human mammalian tissue are more clearly recognised as being of foreign origin by the human host.
  • suitable organs available for transplantation especially since an organ must be removed very shortly after the death of a donor and transplanted whilst the organ is still viable.
  • the necessity for careful tissue typing exacerbates the problem of finding a suitable donor organ for any one individual.
  • xenografts have been successful, with organs from non-primate mammals being transplanted without rejection into humans. In all such cases, however, the transplanted tissues are exceptional in that they do not induce a hyperacute immune response in the host, so that any adverse reaction by the host may be adequately controlled by the use of immunosuppressive drugs.
  • An example of a successful xenograft into humans is the replacement of a human aortic heart valve with the aortic heart valve of a pig.
  • transgenic animals Several uses for transgenic animals have been proposed. For example, in WO-A-88/00239 , it was suggested that the mammary gland of a mammal could be used to produce foreign proteins by the introduction to the animal of a DNA segment comprising the coding sequence of the foreign protein operably linked to the appropriate control sequence of a milk protein gene.
  • xenograft acceptance can be achieved by inclusion of transgenes expressing homologous complement restriction factors (HCRF) .
  • HCRF homologous complement restriction factors
  • Sandrin et al (in Proc . Natl . Acad. Sci. USA __ ⁇ : 11391- 11395 (1993)) also suggested the use of transgenic technology to produce animal organs suitable for transplantation into human bodies. Sandrin et al envisaged that a transgenic animal manipulated to mimic human cells could be used in xenograft procedures.
  • transgenic Typically several hundred copies of the transgene are injected, but only a low proportion of gene transfer operations are successful, especially for large domestic animals such as pigs, goats, sheep and cattle.
  • each cell of the transgenic animal contains a copy of the transgene
  • the animal is a "true” transgenic and will transmit the transgene to its offspring in the normal Mendelian manner.
  • the animal is said to be "mosaic". In a mosaic animal inheritance of the transgene will only occur if the transgene is present in the gametes.
  • Inactivation is believed to result from the spreading of the heterochromatic state along the chromosome and the extent of PEV may vary from cell to cell, resulting in mosaic expression of affected genes. Although it has been most intensively studied in Drosophila, the phenomenon also occurs in yeast (Allshire et al (1994) Cell 25. 157-169) and mammals (Cattanach (1974) Genetic Res 2 291-306) . Current understanding of the underlying mechanisms which control PEV is incomplete.
  • position effect has been used to describe the highly variable and copy number independent expression seen in the analysis of independent lines of transgenic mammals generated using the same genetic construct (for review, see Clark et al (1994) Reprod Fertil Dev. 6 . 589-98) .
  • the transgenes are not only subject to the position effect described above but also to PEV.
  • PEV has been reported in transgenic mouse lines expressing the tyrosinase gene (Mintz & Bradl, (1991) Proc Natl Acad Sci USA fijj.
  • Tyrosinase is a key enzyme of melanin biosynthesis and is responsible for the formation of pigment in the skin and eye melanocytes .
  • PEV effects of tyrosinase expression can be easily monitored when patchy rather than uniform coat colour is visually detected.
  • Transgenic mouse lines constructed with small sections ( 6kb) of the tyrosinase gene often show patchy coat expression, ie PEV. The extent of PEV was found to vary from line to line and also between different generations of the same line (Beerman et al (1990) EMBO J.
  • Tanaka et al (Development 108; 223-227 (1990)) reported the production of transgenic mice expressing the tyr mini-gene.
  • Four transgenic founder mice were produced, and the extent of pigmentation varied between the four transgenic animals.
  • the progeny of one of the founder mice, Tg Tyrs-J3 also had a patchy phenotype and Tanaka et al concluded that "it is unlikely that the different phenotypes of Tg Tyrs-J3 were the result of cellular mosaicism".
  • transgenic ie the progeny of G 0 founder
  • the exogenous DNA inserted into the chromosomes of transgenic animals are expressed in a non-uniform (patchy) fashion within the tissues, even though all cells contain the exogenous DNA and theoretically should be able to express it.
  • the non-uniform expression occurs even when a tissue-specific promoter has been operably linked to the exogenous DNA inserted.
  • Variegated expression is particularly problematic in transgenic animals intended for xenograft donation, where all cells of the xenograft organ must be uniformly protected by expression of the transgene for transplantation to be successful.
  • Non-uniform expression has been reported in the literature, but the importance of this phenomenon for xenograft procedures has not been recognised.
  • the transgenic material is intended for use in transplantation the presence of cells which are recognised as "non-self" by the host's immune system may be sufficient to render the xenograft useless.
  • the non-protected cells will be vulnerable to attack by the immune system and will be destroyed. This will adversely affect the function of the xenograft.
  • the present invention provides a solution to the problem of non-uniform expression in cells of a xenograft organ, which problem has just been recognised for the first time.
  • the present invention therefore provides a transgene construct comprising a protein encoding region operably linked to an expression control region and to substantial portion of an element located in the 5' sequence naturally upstream from the protein encoding region.
  • the element preferably includes a locus control region (LCR) .
  • the construct according to the present invention may conatin two or more protein encoding genes which are of interest; these genes may be operably linked to the same or different expression control sequences and to the same or different 5' sequences. It may be convenient for the construct to have more than one protein encoding regions under the influence of the same 5' region, whilst being operably linked to the same or different expression control sequences.
  • protein encoding region is used herein to refer to a polynucleotide (usually double stranded DNA) which encodes for a polypeptide, protein or a part thereof. Generally expression of the polypeptide, protein or a part thereof in the host cell has a desired effect.
  • the protein encoding region may code for a polypeptide, protein or a part thereof capable of altering the i munogenicity of the host cell in which expression occurs, either by altering an epitome normally present on the host cell surface or the polypeptide, protein or part thereof itself being located on the host cell surface.
  • 5' sequence naturally upstream of said protein encoding region is used herein to refer to the 5' sequence which is upstream of the protein encoding region in its natural position within a genome, ie before genetic manipulation.
  • substantially portion refers to a part of the 5' sequence of over lkb, preferably over 20kb, for example over 50kb in length.
  • substantially portion encompasses functional equivalents of the wild-type sequence, ie it includes deletions, insertions, base substitutions and any other alteration which does not detrimentally affect the function of the 5' region to promote uniform expression of the transgene in all cells where it is present.
  • locus control region (abbreviated to "LCR”) is defined herein as the non-coding polynucleotide naturally present upstream of its associated protein encoding sequence (or a functional equivalent thereof) and which confers the following attributes to said associated protein encoding sequence when operably linked thereto:
  • transgene having positional independence will be able to express the protein encoding portions thereof, at a level which is not significantly influenced by the site of integration of the transgene into the genome of the host cell.
  • the random nature of transgene integration causes a significant variation in the levels of expression obtained from transformed cells. Inclusion of the LCR will obviate these differences in expression levels.
  • LCRs can confer the above properties on any functional gene to which it is operably linked.
  • the expression levels obtained are not a simple multiple related to the number of copies of the transgene. Inclusion of the LCR will also obviate these differences, so that insertion of the expression levels obtained will increase in direct correlation to the number of copies of the transgene integrated into the genome of the test.
  • LCR LCR-like resicleic acid
  • inclusion of the LCR may enable the transgene to achieve expression levels similar to the level of expression normally achieved by the protein encoding region when in its natural environment. This is a significant advantage since the expression levels obtained from a transgene is usually below that observed for the wild-type gene.
  • the LCR and (substantially) all of the naturally intervening sequence located between the LCR and the protein encoding region of the gene of interest is present in the transgene construct.
  • the LCRs and associated gene(s) will be selected for their activity in endothlial cells.
  • the transgene construct is desirably used in creating transgenic material, such as a transgenic animal.
  • the transgenic material forms a further aspect of the present invention. Whilst the transgenic material is generally in the form of a transgenic animal, or an organ (or part of an organ) thereof, in certain situations it may sufficient to transplant other xenograft material such as, for example, transgenic material in cell culture form.
  • transgenic material is present as part of a transgenic animal
  • a transgenic animal such an animal also forms a further aspect of the present invention.
  • Suitable animals include pigs, cattle, goats, sheep as well as primates such as baboons and apes. Pigs are especially preferred.
  • the transgenic material of the invention (usually in the form of transgenic animal or an organ, preferably a whole or substantially whole organ, therefrom) is primarily intended as a xenograft in transplantation operations, generally for transplantations into a human body. Inclusion of the LCR in the transgene construct promotes uniform expression of the protein-encoding region of the transgene construct.
  • the present invention provides the use of the transgene construct (as defined above) to produce transgenic material suitable for xenograft transplantation.
  • the protein encoding region desirably encodes for a protein able to alleviate rejection of the xenograft (especially to alleviate hyperacute rejection of the xenograft) following transplantation into a patient, for example a human patient.
  • the complement cascade is regulated at several stages by various proteins which may either be located at the cell surface or are in the liquid phrase. Mention may be made of CR1, CR2 , CD55 (also called Decay Acceleration Factor or DAF), CD46 (also called MCP), CD59 (also called MRL), C4 binding protein, clusterin and vitronectin.
  • DAF Decay Acceleration Factor
  • MCP also called MCP
  • CD59 also called MRL
  • C4 binding protein clusterin and vitronectin.
  • the present invention provides the use of transgenic material for xenograft transplantation into a human or non-human (preferably mammalian) animal body, said transgenic material comprising cells having a transgene construct incorporated into a chromosome thereof, said construct comprising a protein encoding region operably linked to an expression control sequence and to a substantial portion of an element located in the 5' sequence naturally upstream of said protein encoding region.
  • said element is a locus control region (LCR).
  • the archetypal locus control region is that first determined for the control of human ⁇ globin expression (Grosveld et al (1987) Cell J L 975-985) . These workers identified DNAase hypersensitive sites up to 50kb upstream (4 sites) and 20kb downstream (one site) of the ⁇ globin gene. When the identified sequences were genetically manipulated into a mini locus coupled to the ⁇ globin gene, and used to make transgenic mice, the resulting mice showed copy number dependent, position-independent expression at endogenous levels per gene copy. Later work indicated similar results could be obtained without the 3' sequence (Collis et al (1990) EMBO J. 9. 223-240) .
  • the present invention provides a method of treatment of the human or non- human (preferably mammalian) animal body, said method comprising transplanting into said body transgenic material, said transgenic material being characterised by the presence of cells having a transgene construct incorporated into a chromosome thereof, said transgene construct comprising a protein encoding region operably linked to an expression control sequence and to a substantial portion of the 5' sequence naturally upstream of a protein encoding region, said 5' sequence preferably comprising a locus control region.
  • the present invention provides a method of manufacturing said transgenic material, said method comprising insertion of a transgene construct (said construct comprising a protein-encoding region operably linked to an expression control sequence and to a substantial portion of the 5' sequence naturally upstream thereof) into a chromosome of a host cell.
  • a transgene construct comprising a protein-encoding region operably linked to an expression control sequence and to a substantial portion of the 5' sequence naturally upstream thereof
  • the substantial portion of the 5' sequence will include a locus control region (LCR) for the protein-encoding portion of the transgene construct.
  • LCR locus control region
  • YACs yeast artificial chromosomes
  • the present invention provides the use of YACs to produce transgenic material for xenograft transplantations, such as organ donation.
  • the present invention provides transgenic material for xenograft transplantation, whereby said material exhibits a uniform expression of the transgene in all cells which would otherwise be likely to induce an immune response in the host after transplantation of said material.
  • the transgenic material includes in a chromosome thereof a transgene construct, which construct comprises a protein encoding region operably linked to an expression control sequence and to a substantial 5' portion naturally upstream, and which will desirably contain a locus control region for the transgene in question.
  • the step of engineering and/or cloning the transgene construct is carried out using yeast artificial chromosome (YAC) technology.
  • the first stage in the preparation of a genetic construct according to the invention is the insertion of the gene of interest and appropriate flanking sequences into a yeast artifical chromosome (YAC).
  • YAC yeast artifical chromosome
  • Suitable YAC vectors are also available commercially eg Y-RC16 (ATCC # 68371) .
  • the identified gene of interest and the 5' upstream region thereof, desirably including the LCR and also all of the intervening sequence, is excised, optionally modified (by addition of linkers or exposure to restriction enzymes) and is then ligated into the YAC vector.
  • LCRs are those which are active in endothelial cells (for example are normally active in such cells), especially vascular endothelial cells.
  • the LCRs of CR1, CR2 , CD55, CD46, CD59, C4 binding protein, clusterin, vitronection, functional fragments and combinations thereof may therefore be particularly useful in this regard.
  • the YAC construct is then amplified in a yeast, such as S cerevisiae.
  • Yeast DNA is then isolated and purified, for example by a mixture of centifugation and PFGE on agarose gel; as described by Schedl et al (1992, 1993) ibid.
  • the YAC DNA so isolated may be introduced into a host cell by spheroblast fusion, lipofection, electroporation or micro-injection. Micro-injection into embryonic stem cells is preferred.
  • Transgenic mammals may be obatined by microinjecting several hundred copies of gene construct into one of the two pronuclei in a zygote.
  • Zygotes may be obtained in vivo from the oviducts ( Roschlau et al., Arch Tierz . Berlin 21:3-8 (1988); Roschlau et al., in J. Reprod. Fertil. (suppl 38), Cell Biology of Mammalian Egg Manipulation, ed Greve et al (1989); Hill et al . , Theriogenology 32:222 (1992); Bowen et al. Biol Reprod. 5) .
  • Bovine zygotes must be centrifuged at 15,000 x g for several minutes to displace opaque lipid in order to visualise the pronuclei with phase contrast, No arski or Hoffman interference contrast optics.
  • 2-4 pi of buffer containing several hundred copies of DNA construct are injected into a pronucleus.
  • the transgenes integrate at the zygote stage prior to DNA replication to ensure that every cell in the adult contains the transgene.
  • several "copies" of the transgene linked together linearly, integrate in a single site on a single chromosome. The site of integration is random. Integration probably occurs after the first round of DNA replication, and perhaps as late as the 2- or 4- cell stage (Wall and Seidel, 1992), resulting in animals that are mosaic with respect to the transgene. Indeed, up to 30% of animals in which transgenes are detected in somatic tissues do not transmit the transgenes to their offspring (or transmit to less than the expected 50%).
  • embryos are either transferred directly into the oviducts of recipients or cultured for a few days and transferred to the uterus of recipient females. Confirmation of transgene integration is obtained by Southern blot analysis of tissues sampled from the progeny after birth. Transgene expression is measured by assaying for the gene product in appropriate tissues, for example in milk. Embryo survival after microinjection, transgene integration frequency, frequency of expression and expression level, and frequency of germline transmission vary according to quantity and quality of DNA construct injected, strain of mice used (Brinster et al., Proc. Natl. Acad Sci. USA £1:4438-4442 (1985)) and skill and technique of the operator performing microinjection.
  • Host material containing the transgene is then identified, for example using a selectable marker which was incorporated on the transgene construct.
  • a selectable marker which was incorporated on the transgene construct.
  • PBMCs Peripheral blood ononuclear cells
  • peripheral blood adherent cells or endothelial samples from the transgenic animal according to the invention may be labelled with a dye specific to the transgene (eg Calcein AM (Molecular Probes) for C5b-9) and then challenged.
  • Challenge may be from any molecule likely to stimulate complement mediated rejection reactions.
  • a suitable challenge would be human blood, especially blood from the intended patient.
  • the mixture may then be incubated (eg at 37°C) and any reaction observed following a time period compatible with the timescale of complement mediated rejection, for example 30 to 60 minutes.
  • Dye released due to cellular damage may be assessed by any convenient means, for example using flow-cytometry on a Becton Dickinson FACSort.
  • Transgenic animals according to the invention will show a much lower release of dye than that seen, for example, in the transgenic animals described (Fodor et al (1995), PNAS 9_1 : 11153 - 11157, figure 5a) indicating that the complement reaction is not triggered to the same extent as for the prior art transgenic animals.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Husbandry (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention porte sur l'identification d'un problème relatif à l'expression des transgènes chez des animaux transgéniques qui rend les matériaux transgéniques inutilisables pour des transplantations de xénogreffes. Le problème identifié réside dans l'expression modifiée du transgène qui rend la xénogreffe susceptible d'un rejet hyperaigu après l'implantation. La solution apportée consiste à inclure une région correspondant à une partie substantielle de la région se trouvant naturellement en amont du gène d'intérêt. Cette région comporte de préférence la région de contrôle du locus (LCR) du gène d'intérêt de préférence avec la section intercalaire qui lie naturellement le LCR au gène d'intérêt. Le produit de construction du transgène décrit est donc d'une taille très importante et il est recommandé de le manipuler et de l'amplifier à l'aide de la technique YAC (chromosomes artificiels de levure).
PCT/GB1995/002839 1994-12-06 1995-12-06 Produit de construction de transgenes et son expression amelioree Ceased WO1996017946A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39906/95A AU3990695A (en) 1994-12-06 1995-12-06 Transgene construct and improved expression thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9424550A GB9424550D0 (en) 1994-12-06 1994-12-06 Construct
GB9424550.3 1994-12-06

Publications (1)

Publication Number Publication Date
WO1996017946A1 true WO1996017946A1 (fr) 1996-06-13

Family

ID=10765457

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1995/002839 Ceased WO1996017946A1 (fr) 1994-12-06 1995-12-06 Produit de construction de transgenes et son expression amelioree

Country Status (3)

Country Link
AU (1) AU3990695A (fr)
GB (1) GB9424550D0 (fr)
WO (1) WO1996017946A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998016634A1 (fr) * 1996-10-14 1998-04-23 Biotechnology And Biological Sciences Research Council Expression transgene

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994013273A1 (fr) * 1992-12-08 1994-06-23 The Trustees Of The University Of Pennsylvania Sequences permettant d'effectuer l'expression genique de haut niveau, de maniere dependante du nombre de copies et essentiellement independante de la position, dans des systemes transgeniques et cellulaires

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994013273A1 (fr) * 1992-12-08 1994-06-23 The Trustees Of The University Of Pennsylvania Sequences permettant d'effectuer l'expression genique de haut niveau, de maniere dependante du nombre de copies et essentiellement independante de la position, dans des systemes transgeniques et cellulaires

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CLARK, A.J. ET AL.: "Chromosomal position effects and the modulation of transgene expression", REPRODUCTION, FERTILITY AND DEVELOPMENT, vol. 6, no. 5, pages 589 - 598 *
COZZI, E. ET AL.: "Comparative analysis of human DAF expression in the tissues of transgenic pigs and man", TRANSPLANTATION PROCEEDINGS; PROCEEDINGS OF THE XVTH WORLD CONGRESS OF THE TRANSPLANTATION SOCIETY, 28 August 1994 (1994-08-28) - 2 September 1994 (1994-09-02), KYOTO, JAPAN *
FODOR, W.L. ET AL.: "Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection", PROC.NATL.ACAD.SCI., vol. 91, 8 November 1994 (1994-11-08), USA, pages 11153 - 11157 *
ROSENGARD, A.M. ET AL.: "Tissue expression of human complement inhibitor, decay-accelerating factor, in transgenic pigs", TRANSPLANTATION, vol. 59, no. 9, 15 May 1995 (1995-05-15), pages 1325 - 1333 *
SCHEDL, A. ET AL.: "A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice", NATURE, vol. 362, 18 March 1993 (1993-03-18), pages 258 - 261 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998016634A1 (fr) * 1996-10-14 1998-04-23 Biotechnology And Biological Sciences Research Council Expression transgene

Also Published As

Publication number Publication date
AU3990695A (en) 1996-06-26
GB9424550D0 (en) 1995-01-25

Similar Documents

Publication Publication Date Title
Cho et al. Generation of transgenic mice
US6316692B1 (en) Transfection, storage and transfer of male germ cells for generation of transgenic species and genetic therapies
KR20180091821A (ko) 유전적 상보성에 의한 인간화 car t-세포 및 혈소판의 조작방법
JP2019502400A (ja) キメラ胚補助臓器作製用の組成物及び方法
AU2002220392A1 (en) Mammalian sex selection using genetic modification
Wilmut et al. Basic techniques for transgenesis
US8119785B2 (en) Nucleic acid sequences and homologous recombination vectors for distruption of a Fel D I gene
EP1253823A1 (fr) Regulation du pourcentage des sexes d'une progeniture par ciblage de transgenes sur les chromosomes sexuels
JP2003526326A (ja) 選択可能トランスジェニック幹細胞作成を目的とする雄性生殖細胞の形質移入
WO2000039294A1 (fr) Cellules porcines incapables d'exprimer l'antigene cd40, et destinees a des xenogreffes
US20190045761A1 (en) Inducible disease models methods of making them and use in tissue complementation
US10626417B2 (en) Method of genetically altering and producing allergy free cats
US6498285B1 (en) Methods for producing transgenic pigs by microinjecting a blastomere
Yang et al. Transgenic farm animals: applications in agriculture and biomedicine
WO1996017946A1 (fr) Produit de construction de transgenes et son expression amelioree
JP4845073B2 (ja) 再構築受精卵の作製方法及びそれを用いたトランスジェニック胚の作製方法
US11732273B2 (en) Methods and compositions for in situ germline genome engineering
Brenig et al. Principles of genetic manipulation of livestock
Bader et al. Scientific background
Damert et al. Generation of transgenic mice by pronuclear injection
Pinkert Genetic engineering of farm mammals
AU711144B2 (en) Materials and methods for management of hyperacute rejection in human xenotransplantation
JPH03244332A (ja) 遺伝子移入動物及びその作製方法
Hirsch et al. Tissue-specific KO of ECM proteins
Ladiges et al. Transgenic animals in toxicology

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase