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Method for efficiently producing cloned chicken

A high-efficiency, recessive white-feather chicken technology, applied in the biological field, can solve problems such as limiting the popularization and application of cloning technology, and achieve the effect of greatly promoting production potential and economic benefits, and the method is practical and feasible.

Pending Publication Date: 2021-09-07
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no clear technical solution for poultry at present, which greatly limits the promotion and application of cloning technology in poultry, and further optimization and improvement are needed

Method used

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  • Method for efficiently producing cloned chicken
  • Method for efficiently producing cloned chicken
  • Method for efficiently producing cloned chicken

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Embodiment Construction

[0027] The present invention will be further described below in conjunction with specific embodiments. But the scope of protection of the present invention is not limited to this:

[0028] A method for efficiently producing cloned chickens, comprising the steps of:

[0029] (1) Chicken OCT4, SOX2, NANOG, LIN28 ( OSNL ) Lentiviral overexpression vector infects CEF;

[0030] Inoculate a 24-well plate with 1.5×105 / well black-feathered Langshan chicken CEF cells in good condition, and culture with DMEM containing 10% FBS; when the cell density is expected to be about 60%, add 300 μL serum-free DMEM medium to each well The diluted virus, with the multiplicity of infection as 10, mixed the four viruses at a ratio of 1:1:1:1, and the final concentration of polybrene was 5 ng / mL, and infected fibroblasts; 6 hours after infection, add 5 ng / mL Dilute the virus in serum-free DMEM medium containing 1 mL of polybrene; 24 hours after infection, replace it with DMEM containing 10% FBS a...

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Abstract

The invention relates to a method for efficiently producing a cloned chicken. The method comprises the following steps: (1) constructing a lentivirus overexpression vector of four factors, namely OCT4, SOX2, NANOG and LIN28, and transfecting a black-feather Langshan chicken fibroblast CEF; (2) inducing the Langshan chicken fibroblast CEF transfected with the OSNL into iPSCs (induced pluripotent stem cells) by utilizing an induction culture medium, and carrying out passage and identification on the iPSCs formed by induction; (3) performing induced differentiation on the iPSCs formed by induction by utilizing an induction culture medium to obtain PGC-like, and identifying the PGC-like; (4) collecting PGC-like cells, injecting the PGC-like cells into recessive white feather chick embryo blood vessels which are hatched for 2.5 days by a chick embryo blood vessel injection method, and determining that the PGC-like cells can migrate and homing in the receptor recessive white feather chick by a frozen section method; and (5) continuously hatching the recessive white feather receptor chick embryos injected with the PGC-like cells of the black-feather Langshan chickens until the recessive white feather receptor chick embryos are hatched. The microsatellite technology proves that the black feather offspring is derived from the PGC-like cell of the donor. According to the invention, a large number of cloned chickens having the same genetic characteristics as donors can be produced.

Description

technical field [0001] The invention relates to a method for efficiently producing cloned chickens, belonging to the field of biotechnology. Background technique [0002] Somatic cell cloning technology is widely used in mammals to study cell characteristics, and has great application value in the study of gene structure and function, gene therapy, occurrence and treatment of genetic diseases, and restoration of endangered animal species. However, this technology cannot be implemented on oviparous animals such as birds and amphibians, which greatly limits the development of avian scientific research and production research. Avian PGCs transplantation technology provides an alternative to produce cloned chickens. In 2006, Larval et al. performed chicken allogeneic PGCs transplantation and produced offspring, which proved that this is the only efficient way to achieve somatic cell cloning in poultry. However, the number of primary isolated PGCs is small, which is far from me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/12C12N5/10A01K67/027
CPCC12N15/86C07K14/4702C12N5/0656C12N5/0696A01K67/0271A01K67/0275C12N2740/15043C12N2800/106C12N2510/00C12N2506/1307C12N2500/44C12N2501/235C12N2501/115C12N2501/125C12N2500/32C12N2501/603C12N2501/602C12N2501/605C12N2501/608C12N2501/155C12N2501/11A01K2207/12A01K2207/05A01K2217/15A01K2227/30A01K2267/02
Inventor 周静左其生李碧春
Owner YANGZHOU UNIV
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