WO2020253894A1 - Method of preparing genetically modified poultry resistent to subgroup j avian leukosis virus - Google Patents

Method of preparing genetically modified poultry resistent to subgroup j avian leukosis virus Download PDF

Info

Publication number
WO2020253894A1
WO2020253894A1 PCT/CZ2020/000030 CZ2020000030W WO2020253894A1 WO 2020253894 A1 WO2020253894 A1 WO 2020253894A1 CZ 2020000030 W CZ2020000030 W CZ 2020000030W WO 2020253894 A1 WO2020253894 A1 WO 2020253894A1
Authority
WO
WIPO (PCT)
Prior art keywords
subgroup
alv
poultry
avian leukosis
virus
Prior art date
Application number
PCT/CZ2020/000030
Other languages
French (fr)
Inventor
Jiří Hejnar
Pavel Trefil
Anna Koslová
Jitka Mucksová
Original Assignee
Ústav molekulární genetiky AV ČR, v.v.i.
Biopharm, A.S.
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 Ústav molekulární genetiky AV ČR, v.v.i., Biopharm, A.S. filed Critical Ústav molekulární genetiky AV ČR, v.v.i.
Priority to CN202080044789.4A priority Critical patent/CN115003155A/en
Priority to EP20743052.1A priority patent/EP4075964A1/en
Priority to AU2020297483A priority patent/AU2020297483A1/en
Priority to KR1020227000427A priority patent/KR20220018016A/en
Publication of WO2020253894A1 publication Critical patent/WO2020253894A1/en

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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
    • 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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present invention relates to engineered, permanent, and genetically determined resistance of poultry to a severe poultry disease - avian leukosis, induced by subgroup J ALV virus.
  • Avian leukosis is a neoplastic disease of the haematopoietic system of poultry caused by avian leukosis viruses.
  • avian leukosis viruses ABV of family Retroviridae, most often of subgroups A and B, more recently of subgroup J, and most recently of subgroup K.
  • ALV avian leukosis viruses
  • Infections with exogenous ALV are encountered in the poultry population very frequently (Jurajda,V., 2002), but the incidence of clinical disease is, in general, markedly lower (1 to 5 % of infected animals) than, e.g., in the case of Mark disease.
  • LL lymphoid leukosis
  • other pathological manifestations of ALV including osteopetrosis, sarcomas and related tumours are found in the field sporadically, or even rarely.
  • myelocytomatosis induced by subgroup J ALV, which in the 1990 started to occur in the meat-type breeds and broilers in many countries worldwide, including the Czech Republic.
  • the economic impact of type J ALV is not only reflected in direct loss by death, but mostly in the negative effect on breeding yields (lower weight gain and laying).
  • the immunosuppressive effect of latent infections with lymphoid leukosis exacerbates the course of other infections.
  • the ALV-J virus When infecting the poultry, the ALV-J virus enters the cells via chNHEI receptor. The retrovirus is then transcribed by reverse transcription and integrates as a provirus into the nuclear DNA of the host cell. The proviral DNA is transcribed to viral RNA, which is translated in the cytoplasm and enveloped into newly formed particles. The release of the particles from the infected cells leads to infection spread to permissive cells in the body of the individual, unless the fowls are either resistant, or protected by restriction factors.
  • the chNHEI receptor is a cellular glycoprotein with ion exchange function, which occurs as a homotrimer. Its prominent first extracellular loop contains amino acid residues critical for interaction with ALV-J. Chicken cells devoid of this glycoprotein are not permissive to ALV-J infection. Similarly, deletion of an amino acid, W38, results in resistance to ALV-J, but not to all other ALV subgroups.
  • the above-mentioned problems are removed by the method of preparing genetically modified poultry resistant to the subgroup J avian leucosis virus according to the present invention, which is based on the fact that this poultry carries the W38 deletion introduced into the chNHEI gene using CRISPR/Cas9-mediated homologous recombination, or optionally, carries the W38 deletion introduced using CRISPR/Cas9 and a specific sgRNA, to yield domestic chicken or domestic turkey resistant to subgroup J avian leukosis virus (ALV).
  • the inventors have created genetically modified poultry individuals, where using genome editing of primordial germ cells (PGC), the tryptophan codon at position 38 (W38) in the chNHEI ion exchanger gene has been deleted. Since chNHEI serves as ALV-J receptor and W38 is an amino acid critical for its receptor but not ion exchanger function, this results in induction of full resistance to ALV-J.
  • PPC primordial germ cells
  • W38 is an amino acid critical for its receptor but not ion exchanger function
  • transgenic individuals can be constructed using poultry embryonic cells,‘primordial germ cells’ (PGC), which can be cultured in vitro, genetically manipulated, and then transplanted into the irradiation-sterilized testes of cockerels.
  • PGC poultry embryonic cells
  • the introduced cells restore spermiogenesis, and this may result in progeny carrying the genetic modification identical with the transplanted PGC.
  • PGC poultry embryonic cells
  • the chHNEI receptor induces full resistance to avian leucosis in an in vitro model of chicken fibroblasts.
  • the method of the present invention in fact represents a combination of results of these two above-mentioned Czech patents, creating a genetically modified line of domestic chicken carrying the W38 deletion.
  • This protection is genetically determined in these chickens, and therefore transmissible to further generations.
  • Inclusion of these resistant modified individuals into a breeding programme may transfer the said property to all individuals, thus significantly increasing the quality of the breeding flock, finally resulting in significant economy in production of eggs, or poultry meet.
  • This novel method may have a worldwide impact on the poultry health, and is particularly relevant to domestic chicken and domestic turkey (collectively referred to as poultry)
  • the method of the present invention related to subgroup J avian leukosis refers to the model of genetically modified individuals with permanent resistance to ALV-J infection created by the inventors. It thus represents a route to eradication of this disease not only in China and Southeast Asia, but also in other parts of the world, because there is a risk of reintroduction of new and more difficult to eradicate Chinese ALV-J strains into regions where this disease is not found at present, including Europe and the United States.
  • the Czech patent No. 307285 specifically refers to the sensitivity and resistance to ALV-J infection in domestic chicken. It constitutes the claim for an isolated DNA molecule encoding mutated protein chNHEI , or its fragment, where tryptophan in position 38 has been deleted, and additionally for an isolated DNA molecule encoding mutated chNHEI , or its fragment, where tryptophan in position 38 has been substituted with glycine or glutamate.
  • This patent describes experimental cell-based verification that the NHE1 sequence of domestic chicken containing mutant W38 confers full resistance to ALV-J infection!
  • Fig. 1 shows a diagram of homologous recombination resulting in W38 deletion in the chicken NHE1 gene.
  • Exon 1 contains the gRNA target sequence (middle) with denoted TGG triplet for W38 (yellow) as a restriction site for Cas9 cleavage (scissors).
  • TGG triplet for W38 yellow
  • scissors a restriction site for Cas9 cleavage
  • nucleotides whose synonymous mutation creates the recognition site for Bsal endonuclease, which then serves for detection of the modified allele.
  • sequencessODN single-stranded oligonucleotides
  • Fig. 2 shows a comparison of the sequence of wild-type chNHEI allele (A) and the sequence after deletion of the codon for W38 (B). Only the immediate vicinity of W38 is shown, denoted by an arrow. The sequence is represented as a chromatogram with transcribed both nucleotide and amino acid sequence.
  • Fig. 3 shows GFP positivity after infection of fibroblasts from the embryos of genotypes W38 -/- (top left), W38 +/- (top right) and W38 +/+ (bottom left) measured by flow cytometry.
  • the x axis GFP fluorescence intensity
  • the y axis cell counts. Percentages of GFP-positive cells are shown in the bottom right corner of the histograms. Bottom right, graph summarising percentages of GFP-positive cells in all examined embryos (four for each genotype).
  • Fig. 4 shows viremia quantification in the chickens of genotypes W38 -/-, +/-, and +/+ (marked on the x axis) after infection with reporter vector RCASBP(J)GFP. Viremia was assessed (on the y axis) in relative units (fold) of negative controls (infection by non-specific RCAS-A virus) after quantitative RT PCR.
  • the preparation of genetically modified lines of poultry is based on the derivation of primordial germ cells from chicken embryos at the age of 24 to 96 hours. These cells were cultured from the samples of collected embryonic blood, or from the head part of the embryo, and expanded in vitro.
  • the W38 deletion was introduced into the PGC genome using CRISPR/Cas9 with gRNA specific for the W38 region in the chNHEI gene and the template for homologous recombination containing the W38 region (Fig. 1).
  • the construct encoding CRISPR/Cas9 with the appropriate gRNA and the template for homologous recombination were introduced into PGC by electroporation in the Amaxa system.
  • Embryos of G2 individuals were incubated until day 10 of development and cultures of embryonic fibroblasts were then prepared. The W38 genotype was verified by DNA isolation, PCR and sequencing. Cultures of embryonic fibroblasts were infected with a virus with ALV-J receptor specificity, in particular, with reporter vector RCASBP(J)GFP, which for easy virus detection transduces the reporter gene for green fluorescent protein (GFP). Quantitative assessment of GFP was done using flow cytometry. Embryonic fibroblasts with W38+/+ and W38+/- genotypes showed identical GFP positivity in about 90 % cells, which indicates almost complete virus spread.
  • Chickens of W38 +/+, W38 +/- and W38 -/- genotypes were infected at the age of several days to two months with reporter vector RCASBP(J)GFP, which maintains the ALV-J receptor specificity while being more aggressive than the prototype strains of ALV-J such as, e.g., HPRS103.
  • the normal course of ALV-J infection is manifested by transient viremia, which according to the age of infected individual develops after several days, typically culminates within 10 days and remains one to two weeks after the culmination.
  • RT-qPCR reverse transcriptase quantitative polymerase chain reaction
  • a complementation assay in which a replication-competent introduced virus complements a defective virus that is then secondarily quantified based on the number of focus-forming transformed cells.
  • the second method of detection verifies the presence of biologically active virus, not just the RNA.
  • the material tested was represented by the serum of infected animals collected at two time points, one and two weeks post infection. Using RT-qPCR, all chickens of the W38-/- genotype (5 chickens in total) were tested negative at both time points.
  • the W38 +/+ and W38 +/- genotypes (10 chickens in total) were, with one exception, positive at the later time point; at the first time point of collection, three chickens were negative, which means a slow onset of viremia during the first week and increase of viremia during the second week.
  • the only negative case may mean here unsuccessful infection, e.g., due to defective inoculation.
  • deletion W38 induces perfect resistance to ALV-J when the virus is inoculated to the circulating blood of juvenile individuals. This type of infection best simulates infection in the conditions of home breeding.
  • Table 1 shows verification of viremia in chickens of genotypes W38 -/-, +/- and +/+ (control wild-type genotype) after infection with reporter vector RCASBP(J)GFP. Viremia was determined as a titre of complemented defective virus 16Q and terminal dilution in two serum samples, 6 and 13 days post infection.
  • the novel method of producing genetically modified poultry resistant to subgroup J avian leucosis virus brings a novel, original solution to eradication of avian leukosis ALV of type J and offers a newly created model of genetically modified individuals with permanent resistance to ALV-J infection. Therefore, it represents a totally revolutionary method of eradication of this disease not only in strong poultry farming countries such as China and Southeast Asia, but also in other parts of the world, because this disease poses a potential risk of being reintroduced into regions where its aetiological agent has already been eradicated.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Environmental Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Fodder In General (AREA)

Abstract

The present invention refers to the method of preparing genetically modified poultry resistant to subgroup J avian leukosis virus, which is characterised by the fact that this poultry carries the W38 deletion introduced into the chNHE1 gene using CRISPR/Cas9-mediated homologous recombination, or optionally, carries the W38 deletion introduced using CRISPR/Cas9 and a specific sgRNA, to yield domestic chicken or domestic turkey resistant to subgroup J avian leukosis virus (ALV).

Description

l
Method of preparing genetically modified poultry resistant to subgroup J avian leukosis virus
Technical field
The present invention relates to engineered, permanent, and genetically determined resistance of poultry to a severe poultry disease - avian leukosis, induced by subgroup J ALV virus.
Background
Avian leukosis is a neoplastic disease of the haematopoietic system of poultry caused by avian leukosis viruses.
The aetiology of avian leukosis is associated with avian leukosis viruses (ALV) of family Retroviridae, most often of subgroups A and B, more recently of subgroup J, and most recently of subgroup K. Infections with exogenous ALV are encountered in the poultry population very frequently (Jurajda,V., 2002), but the incidence of clinical disease is, in general, markedly lower (1 to 5 % of infected animals) than, e.g., in the case of Mark disease. Of leukosis forms, the relatively most frequent is lymphoid leukosis (LL); other pathological manifestations of ALV including osteopetrosis, sarcomas and related tumours are found in the field sporadically, or even rarely. An exception is myelocytomatosis, induced by subgroup J ALV, which in the 1990 started to occur in the meat-type breeds and broilers in many countries worldwide, including the Czech Republic. The economic impact of type J ALV is not only reflected in direct loss by death, but mostly in the negative effect on breeding yields (lower weight gain and laying). Moreover, the immunosuppressive effect of latent infections with lymphoid leukosis exacerbates the course of other infections.
When infecting the poultry, the ALV-J virus enters the cells via chNHEI receptor. The retrovirus is then transcribed by reverse transcription and integrates as a provirus into the nuclear DNA of the host cell. The proviral DNA is transcribed to viral RNA, which is translated in the cytoplasm and enveloped into newly formed particles. The release of the particles from the infected cells leads to infection spread to permissive cells in the body of the individual, unless the fowls are either resistant, or protected by restriction factors.
In the retrovirus lifecycle, the sensitivity or resistance to the virus is decided upon by the presence of the appropriate receptor molecule on the surface of the target cell. The chNHEI receptor is a cellular glycoprotein with ion exchange function, which occurs as a homotrimer. Its prominent first extracellular loop contains amino acid residues critical for interaction with ALV-J. Chicken cells devoid of this glycoprotein are not permissive to ALV-J infection. Similarly, deletion of an amino acid, W38, results in resistance to ALV-J, but not to all other ALV subgroups. Deletions or substitutions of W38 have been found in NHE1 of pheasants and most galliform birds; sensitive species thus only include domestic chicken, four wild fowl species, turkey, and several species of New World quails from the genera Calipepla, Colinus a Oreortyx (Plachy J, Reinisova M, Kucerova D, Senigl F, Stepanets V, Hron T, Trejbalova K, Elleder D, Hejnar J.: Identification of New World quails susceptible to infection with avian leukosis virus subgroup J. Journal of Virology 91(3): e02002-16, 2017). According to our research, neither the breeds of domestic hens nor primitive domestic chicken stocks contain alleles with the W38 deletion. As a result, there is no natural source of genetic variability to be used to selectively breed resistant animals (Reinisova M, Plachy J, Kucerova D, Senigl F, Vinkler M, Hejnar J.: Genetic Diversity of NHE1, Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species. PloS One 11 (3):e0150589).
In countries with developed poultry industry, systematic and long-term selection of animals suffering from this disease is used for its eradication (e.g., the last focus of ALV-J infection in the United States was eliminated in 2007 (Malhotra S, Justice J 4th, Lee N, Li Y, Zavala G, Ruano M, Morgan R, Beemon K: Complete genome sequence of an American avian leukosis virus subgroup J isolate that causes hemangiomas and myeloid leukosis. Genome Announcements 3(2): pii: e01586-14, 2015). However, this requires rather a demanding and costly screening of animals during the breeding process, which can only be used in factory farming under full control of infectious diseases. In China, and in fact in the entire Asia and Africa, where poultry is commonly sold at markets at any age and fattening is also practiced at small family farms without any control, it is so far unrealistic to be rid of this disease despite the eradication programmes that have been launched in recent years.
Summary of the invention
The above-mentioned problems are removed by the method of preparing genetically modified poultry resistant to the subgroup J avian leucosis virus according to the present invention, which is based on the fact that this poultry carries the W38 deletion introduced into the chNHEI gene using CRISPR/Cas9-mediated homologous recombination, or optionally, carries the W38 deletion introduced using CRISPR/Cas9 and a specific sgRNA, to yield domestic chicken or domestic turkey resistant to subgroup J avian leukosis virus (ALV).
The inventors have created genetically modified poultry individuals, where using genome editing of primordial germ cells (PGC), the tryptophan codon at position 38 (W38) in the chNHEI ion exchanger gene has been deleted. Since chNHEI serves as ALV-J receptor and W38 is an amino acid critical for its receptor but not ion exchanger function, this results in induction of full resistance to ALV-J. The present invention isjjased on two already granted Czech patents Nos. 307102 and 307285.
According to the Czech patent No. 307102, we are aware that transgenic individuals can be constructed using poultry embryonic cells,‘primordial germ cells’ (PGC), which can be cultured in vitro, genetically manipulated, and then transplanted into the irradiation-sterilized testes of cockerels. The introduced cells restore spermiogenesis, and this may result in progeny carrying the genetic modification identical with the transplanted PGC. According to the Czech patent No. 307285, we are aware that the deletion of amino acid W38 in the chHNEI receptor induces full resistance to avian leucosis in an in vitro model of chicken fibroblasts.
The method of the present invention in fact represents a combination of results of these two above-mentioned Czech patents, creating a genetically modified line of domestic chicken carrying the W38 deletion. By preventing entry of leukosis viruses into the cells of these individuals, permanent resistance is induced. This protection is genetically determined in these chickens, and therefore transmissible to further generations. Inclusion of these resistant modified individuals into a breeding programme may transfer the said property to all individuals, thus significantly increasing the quality of the breeding flock, finally resulting in significant economy in production of eggs, or poultry meet. This novel method may have a worldwide impact on the poultry health, and is particularly relevant to domestic chicken and domestic turkey (collectively referred to as poultry)
The method of the present invention related to subgroup J avian leukosis refers to the model of genetically modified individuals with permanent resistance to ALV-J infection created by the inventors. It thus represents a route to eradication of this disease not only in China and Southeast Asia, but also in other parts of the world, because there is a risk of reintroduction of new and more difficult to eradicate Chinese ALV-J strains into regions where this disease is not found at present, including Europe and the United States.
As was already mentioned, the Czech patent No. 307285 specifically refers to the sensitivity and resistance to ALV-J infection in domestic chicken. It constitutes the claim for an isolated DNA molecule encoding mutated protein chNHEI , or its fragment, where tryptophan in position 38 has been deleted, and additionally for an isolated DNA molecule encoding mutated chNHEI , or its fragment, where tryptophan in position 38 has been substituted with glycine or glutamate. This patent describes experimental cell-based verification that the NHE1 sequence of domestic chicken containing mutant W38 confers full resistance to ALV-J infection! In this patent, the inventors have found that when tryptophan in the chHNEI receptor is substituted with glycine, this substitution causes full resistance to avian leukosis in an in vitro model of chicken fibroblasts. This fact could only be confirmed/rejected by creating transgenic poultry where this deletion or substitution has been introduced into the genome using CRISPR/Cas9 genome editing. For this purpose, the inventors used the newly created model of preparation of transgenic animals according to the Czech patent No. 307102, which is directly related to the method of producing avian sperm with fertilising capacity developed in the testis of a sterilised recipient cockerel after transplantation of donor primordial germ chicken cells, allowing perfectly feasible and practical creation of transgenic individuals with about 50% high efficiency.
Description of drawings
In the appended drawings, Fig. 1 shows a diagram of homologous recombination resulting in W38 deletion in the chicken NHE1 gene. Top, the intron- exon structure of the chNHEI gene. Exon 1 contains the gRNA target sequence (middle) with denoted TGG triplet for W38 (yellow) as a restriction site for Cas9 cleavage (scissors). In red are denoted nucleotides whose synonymous mutation creates the recognition site for Bsal endonuclease, which then serves for detection of the modified allele. Bottom, sequences of single-stranded oligonucleotides (ssODN) to be used as a template for homologous recombination in the chicken and turkey.
Fig. 2 shows a comparison of the sequence of wild-type chNHEI allele (A) and the sequence after deletion of the codon for W38 (B). Only the immediate vicinity of W38 is shown, denoted by an arrow. The sequence is represented as a chromatogram with transcribed both nucleotide and amino acid sequence.
Fig. 3 shows GFP positivity after infection of fibroblasts from the embryos of genotypes W38 -/- (top left), W38 +/- (top right) and W38 +/+ (bottom left) measured by flow cytometry. The x axis, GFP fluorescence intensity, the y axis, cell counts. Percentages of GFP-positive cells are shown in the bottom right corner of the histograms. Bottom right, graph summarising percentages of GFP-positive cells in all examined embryos (four for each genotype).
Fig. 4 shows viremia quantification in the chickens of genotypes W38 -/-, +/-, and +/+ (marked on the x axis) after infection with reporter vector RCASBP(J)GFP. Viremia was assessed (on the y axis) in relative units (fold) of negative controls (infection by non-specific RCAS-A virus) after quantitative RT PCR.
The method of the present invention has been well validated by the inventors in the laboratories of the Institute of Molecular Genetics of the Czech Academy of Sciences, v. v. i., Prague, CZ, and BIOPHARM a.s., Pohofi-Chotoun, CZ. The following examples of embodiments of the present invention are only for illustration, without limiting the invention in any way.
Examples
Example 1
Preparation of a domestic chicken line with modified receptor gene chNHEI
The preparation of genetically modified lines of poultry is based on the derivation of primordial germ cells from chicken embryos at the age of 24 to 96 hours. These cells were cultured from the samples of collected embryonic blood, or from the head part of the embryo, and expanded in vitro. The W38 deletion was introduced into the PGC genome using CRISPR/Cas9 with gRNA specific for the W38 region in the chNHEI gene and the template for homologous recombination containing the W38 region (Fig. 1). The construct encoding CRISPR/Cas9 with the appropriate gRNA and the template for homologous recombination were introduced into PGC by electroporation in the Amaxa system. After electroporation, cells positive for GFP fluorescence associated with CRISPR/Cas9 were selected by sorting of individual cells in a flow cytometer. A well-growing expanded clone with molecularly confirmed W38 deletion was then orthotopically transplanted to sterilised recipient cockerels according to CZ patent No. 307102. Five months later, restored spermiogenesis was observed, and the ejaculate of recipient cockerels was used for artificial insemination of hens, first intramagnally and then intravaginally. The offspring exclusively comprised individuals heterozygous for the W38 deletion. After reaching sexual maturity, the heterozygous animals were crossed and in G2, all genotypes, i.e., W38 +/+, W38 +/- and W38 -/-, segregated in the expected ratio. The accordance with the expected ratio suggests that the W38 deletion does not represent a significant burden for its carrier that would reduce its utility in the breed. The W38 deletion was verified at all levels (PGC clones, ejaculate of recipient cockerels, blood or feather pulp of G1 and G2 individuals) using molecular genetic methods, i.e., DNA isolation, amplification of the specific segment of chNHEI by polymerase chain reaction (PCR) and sequencing of the PCR product. Exemplary DNA sequences are shown in Fig. 2. Example 2
Demonstration of resistance of W38 deletion carriers to ALV-J
The resistance of W38 -/- individuals was demonstrated by three independent methods that are routine in the art of virology research and that have been specifically established for ALV-J.
A. In vitro infection of embryonic fibroblasts
Embryos of G2 individuals were incubated until day 10 of development and cultures of embryonic fibroblasts were then prepared. The W38 genotype was verified by DNA isolation, PCR and sequencing. Cultures of embryonic fibroblasts were infected with a virus with ALV-J receptor specificity, in particular, with reporter vector RCASBP(J)GFP, which for easy virus detection transduces the reporter gene for green fluorescent protein (GFP). Quantitative assessment of GFP was done using flow cytometry. Embryonic fibroblasts with W38+/+ and W38+/- genotypes showed identical GFP positivity in about 90 % cells, which indicates almost complete virus spread. In contrast, embryonic fibroblasts with the W38-/- genotype displayed GFP positivity in less than 0.05 % cells, which represents the natural background given by autofluorescence (Fig. 3). It can be concluded that the W38-/- genotype is perfectly resistant to ALV-J infection at the level of embryonic fibroblasts.
B. In vivo infection of chickens
Chickens of W38 +/+, W38 +/- and W38 -/- genotypes were infected at the age of several days to two months with reporter vector RCASBP(J)GFP, which maintains the ALV-J receptor specificity while being more aggressive than the prototype strains of ALV-J such as, e.g., HPRS103. The normal course of ALV-J infection is manifested by transient viremia, which according to the age of infected individual develops after several days, typically culminates within 10 days and remains one to two weeks after the culmination. The virus presence in vivo was this time verified both molecularly, by quantitative detection of viral genomic RNA using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and biologically, by a complementation assay in which a replication-competent introduced virus complements a defective virus that is then secondarily quantified based on the number of focus-forming transformed cells. The second method of detection verifies the presence of biologically active virus, not just the RNA. In both cases, the material tested was represented by the serum of infected animals collected at two time points, one and two weeks post infection. Using RT-qPCR, all chickens of the W38-/- genotype (5 chickens in total) were tested negative at both time points. The W38 +/+ and W38 +/- genotypes (10 chickens in total) were, with one exception, positive at the later time point; at the first time point of collection, three chickens were negative, which means a slow onset of viremia during the first week and increase of viremia during the second week. The only negative case may mean here unsuccessful infection, e.g., due to defective inoculation. These results are summarised in Fig. 4. In the complementation assay, at the first time point of collection, only sporadic cases of positivity were found in W38 +/+ and W38 +/- genotypes. At the second time point, samples of animals with these genotypes were positive with one exception (the same individual as in the previous experiment), while all W38 -/- genotypes remained negative (Table 1). In summary, we can conclude that deletion W38 induces perfect resistance to ALV-J when the virus is inoculated to the circulating blood of juvenile individuals. This type of infection best simulates infection in the conditions of home breeding.
Table 1
Chicken Chicken Age of chicken Virus titre (lU/ml) genotype number at the time of 6 days p.i. 13 days p.i.
infection (days)
W38 -/- 604 28 0 0 W38 -/- 606 28 0 0 W38 -/- 611 20 0 0 W38 -/- 612 20 0 0 W38 -/- 615 14 0 0
W38 +/- 601 28 0 101 W38 +/- 603 28 0 102 W38 +/- 605 28 0 101 W38 +/- 610 20 102 103 W38 +/- 616 20 0 103 W38 +/- 618 14 0 102 W38 +/- 619 14 101 103
W38 +/+ 617 14 102 103 Table 1 shows verification of viremia in chickens of genotypes W38 -/-, +/- and +/+ (control wild-type genotype) after infection with reporter vector RCASBP(J)GFP. Viremia was determined as a titre of complemented defective virus 16Q and terminal dilution in two serum samples, 6 and 13 days post infection.
C. Tumour induction by ALV-J-pseudotyped virus
To simulate formation of solid tumours after ALV-J infection, we pseudotyped the transforming virus present in quail cell line 16Q and containing the -src oncogene by a virus with ALV-J receptor specificity, namely by the RCASBP(J)GFP vector. This results in acutely transforming virus, with oncogene v-src enveloped with the ALV-J viral glycoprotein, which is capable of inducing fast growing sarcomas in the site of inoculation, in our case in the wing web. We inoculated chickens at the age of 10 days to two months, and the incidence and growth of sarcomas were monitored during one month. All chickens of genotypes W38 +/+ and W38 +/- developed progressively growing sarcomas, whereas chickens of genotype W38 -/- did not develop any tumour in any one of the cases. Tumours in animals of the W38 +/+ genotype grew faster than tumours in chickens of the W38 +/- genotype, which indicates a mild negatively dominant effect of the allele carrying the W38 deletion. This is probably associated with chNHEI trimerization. It can be concluded that this experiment has demonstrated (perfect) full resistance to ALV-J in case of homozygous W38 deletion.
The above-mentioned examples (recently in extenso published in Koslova A, Trefil P, Mucksova J, Reinisova M, Plachy J, Kalina J, Kucerova D, Geryk J, Krchlikova V, Lejckova B, Hejnar J: Precise CRISPR/Cas9 Editing of the NHE1 Gene Renders Chickens Resistant to the J Subgroup of Avian Leukosis Virus. Proc Natl Acad Sci U S A. 117::2108-2112, 2020) conclusively document that the new line of domestic chicken created using the method of the present invention, which carries a homozygous deletion W38 in the chNHEI gene, is perfectly resistant to pathogenic virus ALV-J while preserving its normal growth, reproductive and utility properties, and without any apparent side effects, thus bringing a great economic effect.
Industrial applicability
The novel method of producing genetically modified poultry resistant to subgroup J avian leucosis virus brings a novel, original solution to eradication of avian leukosis ALV of type J and offers a newly created model of genetically modified individuals with permanent resistance to ALV-J infection. Therefore, it represents a totally revolutionary method of eradication of this disease not only in strong poultry farming countries such as China and Southeast Asia, but also in other parts of the world, because this disease poses a potential risk of being reintroduced into regions where its aetiological agent has already been eradicated.
References:
JURAJDA, Vladimir. Nemoci drubeze a ptactva - virove infection. 1. vyd. Brno: Edicni stfedisko VFU. Brno, 2002. 184 s. ISBN 80-7305-436-1.
Malhotra S, Justice J 4th, Lee N, Li Y, Zavala G, Ruano M, Morgan R, Beemon K: Complete genome sequence of an american avian leukosis virus subgroup j isolate that causes hemangiomas and myeloid leukosis. Genome Announcements 3(2): pii: e01586-14, 2015
Plachy J, Reinisova M, Kucerova D, Senigl F, Stepanets V, Hron T, Trejbalova K, Elleder D, Hejnar J.: Identification of New World quails susceptible to infection with avian leukosis virus subgroup J. Journal of Virology 91(3): e02002-16, 2017
Reinisova M, Plachy J, Kucerova D, Senigl F, Vinkler M, Hejnar J.: Genetic Diversity of NHE1 , Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species. PloS One 11(3):e0150589), 2016
Koslova A, Trefil P, Mucksova J, Reinisova M, Plachy J, Kalina J, Kucerova D, Geryk J, Krchlikova V, Lejckova B, Hejnar J: Precise CRISPR/Cas9 Editing of the NHE1 Gene Renders Chickens Resistant to the J Subgroup of Avian Leukosis Virus. Proc Natl Acad Sci U S A. 117::2108-2112, 2020
CZ patent No. 307102
CZ patent No. 307285

Claims

C L A I M S
1. The method of preparing genetically modified poultry resistant to subgroup J avian leukosis virus, which is characterised by the fact that this poultry carries the W38 deletion introduced into the chNHEI gene using CRISPR/Cas9-mediated homologous recombination, or optionally, carries the W38 deletion introduced using CRISPR/Cas9 and a specific sgRNA, to yield domestic chicken or domestic turkey resistant to subgroup J avian leukosis virus (ALV).
PCT/CZ2020/000030 2019-06-19 2020-06-18 Method of preparing genetically modified poultry resistent to subgroup j avian leukosis virus WO2020253894A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202080044789.4A CN115003155A (en) 2019-06-19 2020-06-18 Preparation method of transgenic poultry capable of resisting subgroup J avian leukosis virus
EP20743052.1A EP4075964A1 (en) 2019-06-19 2020-06-18 Method of preparing genetically modified poultry resistent to subgroup j avian leukosis virus
AU2020297483A AU2020297483A1 (en) 2019-06-19 2020-06-18 Method of preparing genetically modified poultry resistent to subgroup J avian leukosis virus
KR1020227000427A KR20220018016A (en) 2019-06-19 2020-06-18 J How to make genetically modified poultry resistant to avian avian leukemia virus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CZPV2019-392 2019-06-19
CZ2019-392A CZ308509B6 (en) 2019-06-19 2019-06-19 Method of preparation modified poultry resistant to avian leukosis virus subgroup J

Publications (1)

Publication Number Publication Date
WO2020253894A1 true WO2020253894A1 (en) 2020-12-24

Family

ID=72660639

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CZ2020/000030 WO2020253894A1 (en) 2019-06-19 2020-06-18 Method of preparing genetically modified poultry resistent to subgroup j avian leukosis virus

Country Status (6)

Country Link
EP (1) EP4075964A1 (en)
KR (1) KR20220018016A (en)
CN (1) CN115003155A (en)
AU (1) AU2020297483A1 (en)
CZ (1) CZ308509B6 (en)
WO (1) WO2020253894A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114410808A (en) * 2022-03-30 2022-04-29 华南农业大学 Genetic resistance molecular marker for avian A, K subgroup avian leukosis and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ307102B6 (en) 2016-06-10 2018-01-10 Biopharm, Výzkumný Ústav Biofarmacie A Veterinárních Léčiv, A.S. A method of production of sperm and transgenic birds
CZ307285B6 (en) 2013-01-28 2018-05-16 Ústav molekulární genetiky AV ČR, v.v.i. Polymorphisms in the strain of NHE1 of the domestic fowl associated with resistance or decreased sensitivity to ALV-J

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101935675B (en) * 2010-01-14 2012-06-06 山东农业大学 Construction and application of cell membrane expression ALV-env fluorescin eukaryotic transgenic expression plasmid
CN102851355B (en) * 2012-03-20 2014-12-10 华南农业大学 Typing method of resistance for resisting subgroup A avian leukosis virus by quality chicken
CN102876675A (en) * 2012-06-20 2013-01-16 韩健宝 Peptide nucleic acid for subgroup J avian leukosis viruses and application of peptide nucleic acid
CN104152445A (en) * 2014-07-30 2014-11-19 华南农业大学 Chicken B,D,E-subgroup avian leukaemia genetic resistance related mononucleotide polymorphism molecular marker and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ307285B6 (en) 2013-01-28 2018-05-16 Ústav molekulární genetiky AV ČR, v.v.i. Polymorphisms in the strain of NHE1 of the domestic fowl associated with resistance or decreased sensitivity to ALV-J
CZ307102B6 (en) 2016-06-10 2018-01-10 Biopharm, Výzkumný Ústav Biofarmacie A Veterinárních Léčiv, A.S. A method of production of sperm and transgenic birds
CZ2016348A3 (en) * 2016-06-10 2018-01-10 Biopharm, Výzkumný Ústav Biofarmacie A Veterinárních Léčiv, A.S. A method of production of sperm and transgenic birds

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ANNA KOSLOVÁ ET AL: "Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells", VIRUSES, vol. 10, no. 11, 2 November 2018 (2018-11-02), pages 605, XP055717468, DOI: 10.3390/v10110605 *
ANNA KOSLOVÁ ET AL: "Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (PNAS), vol. 117, no. 4, 21 January 2020 (2020-01-21), pages 2108 - 2112, XP055728892, ISSN: 0027-8424, DOI: 10.1073/pnas.1913827117 *
D. KUCEROVA ET AL: "Nonconserved Tryptophan 38 of the Cell Surface Receptor for Subgroup J Avian Leukosis Virus Discriminates Sensitive from Resistant Avian Species", JOURNAL OF VIROLOGY, vol. 87, no. 15, August 2013 (2013-08-01), US, pages 8399 - 8407, XP055728874, ISSN: 0022-538X, DOI: 10.1128/JVI.03180-12 *
HONG JO LEE ET AL: "Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J)", DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY., vol. 77, December 2017 (2017-12-01), US, pages 340 - 349, XP055717473, ISSN: 0145-305X, DOI: 10.1016/j.dci.2017.09.006 *
ISAO OISHI ET AL: "Targeted mutagenesis in chicken using CRISPR/Cas9 system", SCIENTIFIC REPORTS, vol. 6, 2016, pages 23980, XP055365536, DOI: 10.1038/srep23980 *
JURAJDA, VLADIMIR, NEMOCI DRUBEZE A PTACTVA - VIROVE INFECTION. 1. VYD. BRNO: EDICNI STREDISKO VFU. BRNO, 2002, pages 184
KOSLOVA ATREFIL PMUCKSOVA JREINISOVA MPLACHY JKALINA JKUCEROVA DGERYK JKRCHLIKOVA VLEJCKOVA B: "Precise CRISPR/Cas9 Editing of the NHE1 Gene Renders Chickens Resistant to the J Subgroup of Avian Leukosis Virus", PROC NATL ACAD SCI USA., vol. 117, 2020, pages 2108 - 2112
MALHOTRA SJUSTICE J 4THLEE NLI YZAVALA GRUANO MMORGAN RBEEMON K: "Complete genome sequence of an American avian leukosis virus subgroup J isolate that causes hemangiomas and myeloid leukosis", GENOME ANNOUNCEMENTS, vol. 3, no. 2, 2015, pages e01586 - 14
PLACHY JREINISOVA MKUCEROVA DSENIGL FSTEPANETS VHRON TTREJBALOVA KELLEDER DHEJNAR J., IDENTIFICATION OF NEW WORLD QUAILS SUSCEPTIBLE TO INFECTION WITH AVIAN LEUKOSIS VIRUS SUBGROUP J. JOURNAL OF VIROLOGY, vol. 91, no. 3, 2017, pages e02002 - 16
PLACHY JREINISOVA MKUCEROVA DSENIGL FSTEPANETS VHRON TTREJBALOVA KELLEDER DHEJNAR J: "Identification of New World quails susceptible to infection with avian leukosis virus subgroup", J. JOURNAL OF VIROLOGY, vol. 91, no. 3, 2017, pages e02002 - 16
REINISOVA MPLACHY JKUCEROVA DSENIGL FVINKLER MHEJNAR J.: "Genetic Diversity of NHE1, Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species", PLOS ONE, vol. 11, no. 3, 2016, pages e0150589

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114410808A (en) * 2022-03-30 2022-04-29 华南农业大学 Genetic resistance molecular marker for avian A, K subgroup avian leukosis and application thereof
CN114410808B (en) * 2022-03-30 2022-07-01 华南农业大学 Genetic resistance molecular marker for avian A, K subgroup avian leukosis and application thereof

Also Published As

Publication number Publication date
CN115003155A (en) 2022-09-02
CZ2019392A3 (en) 2020-10-07
AU2020297483A1 (en) 2021-12-23
EP4075964A1 (en) 2022-10-26
CZ308509B6 (en) 2020-10-07
KR20220018016A (en) 2022-02-14

Similar Documents

Publication Publication Date Title
Rubin Genetic control of cellular susceptibility to pseudotypes of Rous sarcoma virus
CA3007066A1 (en) Methods for gender determination of avian embryos in unhatched eggs and means thereof
JP6644276B2 (en) Method of genetically modifying poultry primordial germ cells, method of producing genetically modified poultry primordial germ cells, method of producing genetically modified poultry, and poultry egg
JP6923232B2 (en) Genetically modified poultry eggs
JP2010029219A (en) Method for developing animal model
Hocking Biology of breeding poultry
WO2020109780A2 (en) Polypeptide and uses thereof
BR112021000989A2 (en) a method to generate sterile progeny
EP0424044A1 (en) Transgenic fowl expressing bovine growth hormone
WO2020253894A1 (en) Method of preparing genetically modified poultry resistent to subgroup j avian leukosis virus
LEVIN et al. Molecular Analysis of Endogenous Virus ev 21-Slow Feathering Complex of Chickens.: 1. Cloning of Proviral-Cell Junction Fragment and Unoccupied Integration Site
US20230313319A1 (en) Molecular marker for genetic resistance of chicken to infection by subgroups a and k avian leukosis virus and use thereof
Doran et al. Genome editing in poultry-opportunities and impacts
CN116411087B (en) Avian leukosis genetic resistance molecular marker tvb for chicken subgroup B3215-3216insCCAnd applications thereof
JPH03210186A (en) Vector for producing transformation poultry
Nassar Genetic diversity of the major histocompatibility complex by using LEI0258 microsatellite marker associated with productive performance and viral diseases in broiler breeders
DK202170116A1 (en) Genetically modified salmon which produce sterile offspring
CN109414494A (en) The method for generating virus
Crittenden et al. Genetic engineering to improve resistance to viral diseases of poultry: a model for application to livestock improvement
AU2020101720A4 (en) An application of an exogenous RIG-I gene in preparation of chicken anti-Newcastle disease virus products
CN111454908A (en) Tpl2 defective MDCK cell strain and construction method and application thereof
CN111849977B (en) Method for preparing transgenic animals by sperm vector, sgRNA for preparing short and small transgenic chickens and preparation method
Salter et al. Gene insertion into the avian germline
Dekkers et al. Genetic Parameters and Genome-Wide Association Study of Newcastle Disease Response Traits in Tanzania and Ghana Local Chicken Ecotypes
USH1065H (en) Transgenic avian line resistant to avian leukosis virus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20743052

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020297483

Country of ref document: AU

Date of ref document: 20200618

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20227000427

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020743052

Country of ref document: EP

Effective date: 20220119

122 Ep: pct application non-entry in european phase

Ref document number: 20743052

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020743052

Country of ref document: EP

Effective date: 20220119