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WO1990011355A1 - Introduction d'un gene exogene dans des oiseaux - Google Patents

Introduction d'un gene exogene dans des oiseaux Download PDF

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Publication number
WO1990011355A1
WO1990011355A1 PCT/GB1990/000388 GB9000388W WO9011355A1 WO 1990011355 A1 WO1990011355 A1 WO 1990011355A1 GB 9000388 W GB9000388 W GB 9000388W WO 9011355 A1 WO9011355 A1 WO 9011355A1
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WO
WIPO (PCT)
Prior art keywords
germ cells
primordial germ
foreign nucleic
embryo
nucleic acid
Prior art date
Application number
PCT/GB1990/000388
Other languages
English (en)
Inventor
Kenneth Simkiss
Original Assignee
National Research Development Corporation
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 National Research Development Corporation filed Critical National Research Development Corporation
Priority to AU52879/90A priority Critical patent/AU648502B2/en
Publication of WO1990011355A1 publication Critical patent/WO1990011355A1/fr

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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

Definitions

  • This invention is in the field of transgenlc animals.
  • foreign nucleic acid especially foreign gene(s)
  • primordial germ cells derived from the embryo of a donor bird that the cells carrying the foreign nucleic acid can be introduced Into the embryo of a recipient bird, and that the foreign nucleic acid is thereby carried Into the germ cells of the embryo and therefore into the germ line of a bird which will be produced from the embryo.
  • transgenlc birds especially poultry and game birds, can be produced.
  • the invention herein can be expressed as a method of introducing foreign nucleic acid, especially a foreign gene, into birds, which comprises providing in vitro foreign nucleic acid which it is desired to introduce into the germ line of a bird, introducing the foreign nucleic acid into explanted primordial germ cells of a bird and then introducing these primordial germ cells into the blood system of a recipient embryo of an incubated egg, at a stage of embryonic development at which Introduced primordial germ cells will settle in the germinal ridge.
  • Figure 1 shows a chicken embryo in surface view
  • Figure 2 is a graph of concentration of primordial germ cells in blood plotted against time (stage of embryonic development);
  • Figure 3 is a transverse section of part of a chick embryo showing the germinal ridge
  • Figures 4 and 5 show proviral DNA constructs used to prepare a replication-defective retroviral vector carrying a foreign gene, this vector being used to infect donor primordial germ cells.
  • PGCs Primordial germ cells
  • primordial germ cells 2 in the germinal crescent 3.
  • the germinal crescent lies between the area opaca and area pellucida anterior to the head of the developing embryo. Also shown are blood vessels 4 and the gonad 5 of the donor embryo.
  • the PGCs migrate from this site of origin, via the bloodstream, to the site of the future gonad, which Is called the germinal ridge. In the embryo of the domestic fowl, this migration occurs after about 50h of incubation at stage 16 of development ( Figure 2: “A”). It is seen as a large pulse of transient primordial germ cells among the normal erythrocytes of the blood. Within a few hours they disappear from the blood and settle in the germinal ridge ( Figure 2: “B”). Figure 2 shows the population of PGCs in the blood stream of the embryo plotted against somite number, which represents a stage of embryonic development. (The somite is a block of muscle. By counting these somites in the head to tail direction of the embryo, its development can be quantised).
  • Figure 3 is a transverse section of a part of the embryo showing the germinal ridge.
  • 11 mesoderm
  • 12 spinal cord
  • 13 extra-embryonic cavity
  • 14 blood vessel
  • 15 PGCs carried in the bloodstream
  • 16 germinal ridge.
  • the PGCs proliferate to form germ cells.
  • the number settling is of the order of a few hundred, while the number of proliferated germ cells produced is of the order of a million in the female. Only a fraction of these germ cells (a few thousand) are carried through to the adult bird, there being extensive atrophy of these oocytes after hatching.
  • Virtually all domestic fowl contain DNA copies of retroviral genomes that have entered the germ line during evolution but are inherited as structural genes.
  • such individuals have been used as donors to inject primordial germ cells Into a "line zero" strain of retrovirus-free birds.
  • Blood, containing PGCs was taken from an embryo of a donor bird and injected into the heart of a recipient embryo. After further incubation, the recipient embryos were dissected and DNA extracted from the gonads, heart, liver and muscle. Untreated donor and recipient embryos were also dissected to provide positive and negative controls. Dot blots and Southern blots were prepared and probed with labelled retroviral DNA. In 4 out of 11 transfer experiments the gonads were found to be labelled with donor cell DNA. In one case the heart was also positive but in other cases the transfer was specific for the gonad tissues.
  • the PGCs are preferred to take the PGCs from the blood stream of the donor embryo, or from a germinal crescent, in order most closely to simulate the natural processes. There appears to be some chemical signal which causes the germ cells to settle in the germinal ridge and it is not certain whether very immature PGCs would have the means of recognising or following the signal.
  • the PGCs will normally be explanted, and possibly then cultured in vitro. In order to prevent them from maturing and differentiating too rapidly, before the foreign nucleic acid can be introduced and the PGCs inoculated into the recipient embryo, it will probably be advisable to introduce Differentiation Inhibitory Activity factor into the culture : see Example 1 for references .
  • the nucleic acid can then be introduced into the PGCs in vitro in any of the now well accepted ways for introducing nucleic acid into cells, e.g. by calcium phosphate transfection of DNA, direct inoculation of DNA into the PGCs or Infection of the culture with a retroviral vector carrying nucleic acid foreign to the bird.
  • the usual techniques apply.
  • the direct inoculation technique is similar to that used for inoculating a pronucleus in conventional transgenic technology.
  • a retroviral vector is used.
  • the vector is preferably one which is not capable of undergoing replication In the PGCs and will normally be formed from two or more DNA constructs which, when acting together, allow integration of the foreign nucleic acid into the germ cell chromosomes.
  • the explanted PGCs will be isolated from surrounding tissue, it is also possible to introduce the foreign nucleic acid Into a sample of PGC-contalning tissue, such as blood of the donor embryo (at a stage of development at which PGCs will be present).
  • a sample of PGC-contalning tissue such as blood of the donor embryo (at a stage of development at which PGCs will be present).
  • the blood can be Infected with an appropriate retroviral vector carrying the foreign nucleic acid.
  • the nucleic acid transferred can be RNA or DNA;
  • DNA can be genomic or cDNA produced from genomic or mRNA. It can be a polynucleotide coding for a polypeptlde. In all cases, appropriate promoters, signal and transcription-termination sequences may be required, as is recognised in the art.
  • the gene introduced may be any of those familiar to those skilled in the poultry genetics field, including growth genes, genes which may impart resistance to poultry diseases such as coccidiosis, Marek's Disease Virus, Newcastle Disease Virus, Infectious Bronchitis Virus, Infectious Bursal Disease Virus and so forth. Further, genes coding for attenuated strains of Salmonella may be of value.
  • the competition between native PGCs of the recipient embryo and the donor PGCs containing the foreign nucleic add for lodgement in the germinal ridge should be altered in favour of the donor PGCs.
  • One method of achieving this is simply to reduce the ratio of native to introduced PGCs.
  • the germinal crescent PGCs of the recipient embryo may be removed by cauterisation or by selective irradiation or by use of a chemosterilant such as the drug Busulphan.
  • the donor PGCs could be cultured and introduced in such large numbers as to swamp the native PGCs.
  • the invention Includes all conventional further steps downstream of the introduction of the foreign nucleic acid to the recipient embryo, such as hatching chicks, rearing birds from these chicks, using the eggs of reared egg-laying birds as a source of genes for a further gene transfer and so on.
  • hatching chicks denotes the newly hatched offspring of any bird, not necessarily of a fowl unless the context so requires.
  • the invention further includes "indirect products of the method” viz. birds which are made transgenic or carry foreign nucleic acid in their germ line through application of the process of the invention to their mother or to any maternal ancestor of the bird's mother or father.
  • the invention is of significance chiefly for gene transfer using cells of the same species or even strain and preferably in poultry such as chickens, ducks, turkeys, geese and guinea fowl or game birds such as pheasants, grouse and partridges.
  • Section A This Example demonstrates in section A that retrovirally infected PGCs can be transferred to retrovirus-free eggs, producing a retrovirally infected embryo.
  • Section B indicates how advantage is taken of this finding to introduce a foreign gene into differentiated tissue of the embryo.
  • a needle and tube were prepared to collect the blood. This consists of a 30 gauge dental needle attached to a 30 cm length of canula tubing ending in a mouth piece. The needle was dipped into heparin (anti-coagulant) solution before use.
  • a donor egg was positioned beneath a binocular microscope with fibre optic illumination and the embryo exposed by removing the shell over the blunt end (air space) of the egg.
  • the needle was inserted into a large vein in the donor embryo, usually the one running down the back of the embryo. Alternatively any other vein or the heart may be used. About 10 ⁇ l of blood, typically containing about 40 PGCs, were drawn into the needle by gentle suction. Alternatively a small syringe may be used to suck the blood into the tube.
  • the "recipient” embryo was derived from a retrovirus-free White Leghorn strain. Such a strain is kept at the Institute for Animal Health, Houghton Laboratory, Houghton, Huntingdon PE17 2DA, England. Eggs from this strain are freely available for sale (and have been sold). An alternative source of supply of retrovirus-free eggs is the USDA station at East Lansing, Michigan, USA.
  • the "recipient” egg was laid on Its side and Its air space pierced with a pair of fine forceps. The egg was wiped with 70% alcohol and a small hole (1 cm 2 ) cut in the centre of the shell with a sterile hacksaw blade. The shell and shell membranes were removed with fine forceps. At this stage, the embryo drops away from the shell as the contents displace the air from the air space.
  • the recipient embryo was located and the blood injected directly into the heart or into the region around the heart or a major blood vessel.
  • the egg was i ncubated i n a normal i ncubator and candled regularly to check growth.
  • the blots were probed with radiolabelled retroviral DNA.
  • the retroviral DNA actually used was from a plasmid designated pRAV-1 used at the Institute for Animal Health, Houghton (see above), but originally a gift from Paula Enrietta of the Imperial Cancer Research Foundation, London. It is available from the Institute, but it is not necessary to use this specific probe. Any of the well known avian retroviral DNA clones will do. (To check that a particular retroviral DNA is suitable is a simple matter, as it lights up certain distinctive bands when DNA taken from avian red blood cells is Southern-blotted and probed with radiolabelled retroviral DNA). The nitrocellulose filters were hybridised overnight and washed at high stringency (65°C, 0.1% SSC). In 4 out of 11 experiments the retroviral DNA was found in the gonad tissue.
  • the explanted primordial germ cells would be maintained in an undifferentiated state using developmental inhibitory activity factor, R.L. Williams et al., Nature 3 36 , 684 - 687 (1988), A.G. Smith et al. , Nature 1988, 336, 688-685 (1988)
  • Gene insertion into the cells would be by conventional methods (Calcium phosphate incorporation; DNA injection or defective retroviral infection).
  • Manipulated primordial germ cells would be inserted into embryos as in steps A5-A8 except that these embryos would have had their germinal crescent cells destroyed (by cautery, irradiation or chemosterilizatlon) or, alternatively, excessive donor PGCs would be given before the mobilization of the endogenous PGCs of the recipient embryo.
  • primordial germ cells would be introduced into sterilized embryos leading to a permanent modification to the germ line of the bird.
  • This Example demonstrates the infection of PGCs either in vitro or in a donor embryo, with a retroviral vector carrying a foreign gene, transferring the infected PGCs to retrovirus-free eggs and detecting the foreign gene in differentiated tissue of the embryo.
  • a replication-defective retroviral vector was prepared from a wild type reticuloendothelial virus, spleen necrosis virus (SNV) as follows.
  • the American Type Culture Collection (ATCC) supplied two spleen necrosis virus (SNV) REVs under accession numbers ATCC 45012 and 45013.
  • ATCC 45013 had a deleted packaging signal.
  • the vector included a proviral DNA helper construct having gag, pol and env genes of
  • SNV SNV, but lacking a packaging signal, whereby the native retroviral genes cannot be packaged. It does not possess a long terminal repeat (LTR) at either end.
  • LTR long terminal repeat
  • An SV40 late promoter is provided. This proviral DNA construct is shown in Figure 4.
  • the other part of the vector was a construct of DNA made from the E. coll. lacZ (beta-galactosidase) gene and the packaging signal and env gene of the SNV, as shown in Figure 5. (The sequences from pBR322, a standard commercially available plasmid, are of no relevance).
  • the vector DNA (both constructs) was co-infected into a quail cell line "QT 35".
  • a quail cell line was used because ordinary chicken cell lines are infected with avian leukemia virus, which is another retrovirus, which might introduce an unwanted infectious virus.
  • avian leukemia virus which is another retrovirus, which might introduce an unwanted infectious virus.
  • a chemical "Lipofectln” (commercially available) was added to the vector DNA.
  • Chicken eggs were incubated to stage 14 of development (roughly 50h). They were swabbed with alcohol and opened in a dish of saline (9g NaCl /litre of water) at 37oC. The embryo was positioned under a hole in a piece of filter paper, cut free from the surrounding tissue and lifted off from the yolk. This left the embryo stretched across a hole in the filter paper. The PGCs in the germinal crescent to the anterior and sides of the embryo could easily be seen and the tissue containing them was then cut out with fine scissors. Each piece of germinal crescent tissue was placed in a vial with approx.
  • Such blood samples normally contain circulating
  • the sample was therefore exposed to 10 ⁇ l of the defective retrovirus for 1 ⁇ 2 to 1h and a sample of 10 ⁇ l taken for injection into a recipient embryo.
  • Recipient embryos were incubated to approximately stage 15 of development.
  • the shell was swabbed with alcohol and the embryo exposed through a hole roughly 1 to 2 cm 2 .
  • a suspension of the retrovirally infected germinal crescent cells (roughly 100 PGCs) in 10 ⁇ l saline was Injected Into the heart of the embryo by cardiac puncture using a fine glass needle. The shell was sealed with tape and returned to the incubator.
  • gonads of embryos killed at 18d were examined by Southern blotting (without prior PCR). Samples of DNA were extracted from the gonads, the DNA cut with restriction endonucl eases and run on an agarose gel. The gel was then probed for retrovirus and positive hybridisation detected.

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Abstract

Procédé permettant d'introduire un acide nucléique étranger, notamment un gène, dans des oiseaux, par exemple des poulets, qui tire profit de l'étape de développement de l'embryon, dans laquelle les cellules germinales primordiales (PGC) se rassemblent dans le croissant germinal, migrent dans le flux sanguin et se fixent dans le bord germinal qui devient la gonade. Le procédé en question consiste à produire in vitro l'acide nucléique étranger que l'on veut introduire dans la ligne germinale d'un oiseau, à introduire ledit acide nucléique étranger dans les cellules germinales primordiales en explant d'un oiseau et à réintroduire ces cellules germinales primordiales dans le système sanguin d'un embryon hôte se trouvant, dans un ÷uf couvé, au stade de développement embryonnaire dans lequel les cellules germinales primordiales se fixent dans le bord germinal.
PCT/GB1990/000388 1989-03-17 1990-03-15 Introduction d'un gene exogene dans des oiseaux WO1990011355A1 (fr)

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AU52879/90A AU648502B2 (en) 1989-03-17 1990-03-15 Introducing an exogenous gene into birds

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GB898906214A GB8906214D0 (en) 1989-03-17 1989-03-17 Introducing an exogenous gene into birds
GB8906214.5 1989-03-17

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JP (1) JPH04504056A (fr)
AU (1) AU648502B2 (fr)
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0710439A3 (fr) * 1994-10-31 1996-05-15 Director Of National Institute Of Animal Industry, Ministry Of Agriculture, Forestry And Fisheries Procédé de cryoconservation de cellules germinales primordiales et cellules germinales
WO1999009817A1 (fr) * 1997-08-22 1999-03-04 Biotechnology And Biological Sciences Research Council Utilisation de l'element mariner pour creer des animaux transgeniques
WO2002067669A3 (fr) * 2001-02-16 2003-02-27 Tranxenogen Inc Production d une lignee germinale d'oiseaux fondee sur une cellule germinale primordiale
EP1050586A4 (fr) * 1998-01-28 2003-07-23 Takara Bio Inc Procede relatif au transfert de gene dans des cellules germinales
US6730822B1 (en) 1997-10-16 2004-05-04 Avigenics, Inc. Vectors in avian transgenesis
US7049480B1 (en) 2000-09-01 2006-05-23 Avigenics, Inc. Methods of enucleating an avian oocyte or zygote using two-photon laser scanning microscopy
US7129390B2 (en) 1997-10-16 2006-10-31 Avigenics, Inc Poultry Derived Glycosylated Interferon Alpha 2b
US7312374B2 (en) 2001-09-18 2007-12-25 Avigenics, Inc Production of a transgenic avian by cytoplasmic injection
US7339090B2 (en) 2001-02-13 2008-03-04 Avigenics, Inc. Microinjection devices and methods of use
US7381712B2 (en) 2003-05-09 2008-06-03 Avigenics, Inc. In vivo transfection in avians
US7511120B2 (en) 1997-10-16 2009-03-31 Synageva Biopharma Corp. Glycosylated G-CSF obtained from a transgenic chicken
US7534929B2 (en) 1997-10-16 2009-05-19 Synageva Biopharma Corp. Avians expressing heterologous protein
US7550650B2 (en) 2001-09-18 2009-06-23 Synageva Biopharma Corp. Production of a transgenic avian by cytoplasmic injection
US7795496B2 (en) 2003-05-09 2010-09-14 Synageva Biopharma Corp. In vivo transfection in avians
US7803362B2 (en) 2003-01-24 2010-09-28 Synageva Biopharma Corp. Glycosylated interferon alpha
US7812127B2 (en) 2006-03-17 2010-10-12 Synageva Biopharma Corp. Glycosylated human G-CSF
US8124732B2 (en) 2005-06-24 2012-02-28 Synageva Biopharma Corp. Composition comprising isolated human CTLA4-Fc fusion protein produced in a transgenic chicken
US8222032B2 (en) 2003-10-07 2012-07-17 Synageva Biopharma Corp. Cell lines and methods for producing proteins
US8563803B2 (en) 1997-10-16 2013-10-22 Synageva Biopharma Corp. Method of making protein in an egg of a transgenic chicken
WO2016091272A1 (fr) * 2014-12-12 2016-06-16 Central Veterinary Research Laboratory Système et procédé pour l'établissement d'une culture à long terme de cellules germinales primordiales aviaires et utilisations associées
US10897881B2 (en) 2012-04-20 2021-01-26 Commonwealth Scientific And Industrial Research Organisation Method of making a chicken with germ cells expressing marker protein

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US7117814B2 (en) * 2001-09-26 2006-10-10 Ovagen International Limited Method of producing avian eggs and birds of germ-free status

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GB8823869D0 (en) * 1988-10-12 1988-11-16 Medical Res Council Production of antibodies

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Science, Volume 243, No. 4890, 27 January 1989, (Washington, DC, US), R.A. BOSSELMAN et al.: "Germline Transmission of Exogenous Genes in the Chicken", pages 533-535 see the whole article *
These de Doctorat, Universite Claude Bernard LYON I, No. 4188, 28 April 1988, F.CHAMBONNET: "Utilisation de Vecteurs Retroviraux Dereives de l'Aev (Avian Erythroblasrtosis Virus) Pour le Transfert de Genes in Vivo Chez le Poulet", see chapter 2.5.1, and figure 2.3 *
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Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0710439A3 (fr) * 1994-10-31 1996-05-15 Director Of National Institute Of Animal Industry, Ministry Of Agriculture, Forestry And Fisheries Procédé de cryoconservation de cellules germinales primordiales et cellules germinales
US5759763A (en) * 1994-10-31 1998-06-02 Director Of National Institute Of Animal Industry, Ministry Of Agriculture, Forestry And Fisheries Method for cryopreservation of primordial germ cells and germ cells
WO1999009817A1 (fr) * 1997-08-22 1999-03-04 Biotechnology And Biological Sciences Research Council Utilisation de l'element mariner pour creer des animaux transgeniques
US8372956B2 (en) 1997-10-16 2013-02-12 Synageva Biopharma Corp. Antibodies produced in a transgenic avian egg white
US8563803B2 (en) 1997-10-16 2013-10-22 Synageva Biopharma Corp. Method of making protein in an egg of a transgenic chicken
US6730822B1 (en) 1997-10-16 2004-05-04 Avigenics, Inc. Vectors in avian transgenesis
US7875762B2 (en) 1997-10-16 2011-01-25 Synageva Biopharma Corp. Method of producing an exogenous protein in the egg of a chicken
US7129390B2 (en) 1997-10-16 2006-10-31 Avigenics, Inc Poultry Derived Glycosylated Interferon Alpha 2b
US8507749B2 (en) 1997-10-16 2013-08-13 Synageva Biopharma Corp. Transgenic chickens producing exogenous protein in eggs
US8519214B2 (en) 1997-10-16 2013-08-27 Synageva Biopharma Corp. Production of exogenous proteins in egg whites of transgenic chickens
US7534929B2 (en) 1997-10-16 2009-05-19 Synageva Biopharma Corp. Avians expressing heterologous protein
US7511120B2 (en) 1997-10-16 2009-03-31 Synageva Biopharma Corp. Glycosylated G-CSF obtained from a transgenic chicken
US7521591B2 (en) 1997-10-16 2009-04-21 Synageva Biopharm Corp. Transgenic chickens that lay eggs containing exogenous proteins
EP1050586A4 (fr) * 1998-01-28 2003-07-23 Takara Bio Inc Procede relatif au transfert de gene dans des cellules germinales
US7049480B1 (en) 2000-09-01 2006-05-23 Avigenics, Inc. Methods of enucleating an avian oocyte or zygote using two-photon laser scanning microscopy
US7339090B2 (en) 2001-02-13 2008-03-04 Avigenics, Inc. Microinjection devices and methods of use
WO2002067669A3 (fr) * 2001-02-16 2003-02-27 Tranxenogen Inc Production d une lignee germinale d'oiseaux fondee sur une cellule germinale primordiale
US7550650B2 (en) 2001-09-18 2009-06-23 Synageva Biopharma Corp. Production of a transgenic avian by cytoplasmic injection
US7312374B2 (en) 2001-09-18 2007-12-25 Avigenics, Inc Production of a transgenic avian by cytoplasmic injection
US7803362B2 (en) 2003-01-24 2010-09-28 Synageva Biopharma Corp. Glycosylated interferon alpha
US7795496B2 (en) 2003-05-09 2010-09-14 Synageva Biopharma Corp. In vivo transfection in avians
US7381712B2 (en) 2003-05-09 2008-06-03 Avigenics, Inc. In vivo transfection in avians
US8222032B2 (en) 2003-10-07 2012-07-17 Synageva Biopharma Corp. Cell lines and methods for producing proteins
US8124732B2 (en) 2005-06-24 2012-02-28 Synageva Biopharma Corp. Composition comprising isolated human CTLA4-Fc fusion protein produced in a transgenic chicken
US7812127B2 (en) 2006-03-17 2010-10-12 Synageva Biopharma Corp. Glycosylated human G-CSF
US10897881B2 (en) 2012-04-20 2021-01-26 Commonwealth Scientific And Industrial Research Organisation Method of making a chicken with germ cells expressing marker protein
US11369096B2 (en) 2012-04-20 2022-06-28 Commonwealth Scientific And Industrial Research Organisation Process for using crispr to transfect primordial germ cells in avians
US12232488B2 (en) 2012-04-20 2025-02-25 Commonwealth Scientific And Industrial Research Organisation Process of transfecting primordial germ cells in an avian
WO2016091272A1 (fr) * 2014-12-12 2016-06-16 Central Veterinary Research Laboratory Système et procédé pour l'établissement d'une culture à long terme de cellules germinales primordiales aviaires et utilisations associées
WO2016091385A1 (fr) * 2014-12-12 2016-06-16 Central Veterinary Research Laboratory Système et procédé pour l'établissement d'une culture à long terme de cellules germinales primordiales aviaires et utilisations associées

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Publication number Publication date
GB2229440A (en) 1990-09-26
CA2050925A1 (fr) 1990-09-18
GB8906214D0 (en) 1989-05-04
EP0463047A1 (fr) 1992-01-02
JPH04504056A (ja) 1992-07-23
AU648502B2 (en) 1994-04-28
AU5287990A (en) 1990-10-22
GB9005883D0 (en) 1990-05-09

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