Method for obtaining cloned pigs with modified genes through continuous cloning

A technology of gene modification and sow, which is applied in the field of pig cloning, can solve the problems of difficulty in achieving the expected effect, low efficiency of individual pigs, narrow application range, etc., and achieve the effect of overcoming the low efficiency of modification

Inactive Publication Date: 2016-07-13
魏红江
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Problems solved by technology

[0002] In recent years, the in-depth research and construction of genetically modified pigs as human disease models has not yet been comprehensive, and there are shortcomings such as low construction efficiency and narrow application range. Animal disease models established from the perspective of animal genetically modified cloned pigs are conducive to in-depth exploration of human-related diseases It has important reference value for the construction of clinical models of human diseases and clinical transformation applications, especially for the study of genetically modified cloned pigs obtained by serial cloning methods as animal disease models. The repeatability of the source, the reliability of the research and the stability of the genetics, the pathological environment, physiological conditions, biochemical mechanism and the mechanism of the occurrence and development of the disease in pigs are very close to those of humans. The genetically modified clones established by this method The pig model provides an important model reference for the study of human-related diseases
[0003] At present, although the pig gene targeting technology can change the specific gene expression pattern of animals, it is extremely difficult to operate. Although the pig transgenic technology is simple to operate and the cycle is short, the efficiency of obtaining individual pigs modified with the target gene is low, and there are large differences among individuals. , it is difficult to achieve the desired effect

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  • Method for obtaining cloned pigs with modified genes through continuous cloning

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Such as figure 1 As shown, a method for obtaining genetically modified cloned pigs through continuous cloning, the specific operation process is carried out in the following steps:

[0028] The first step, the culture of genetically modified porcine fibroblast donor cells and the establishment of cell lines

[0029] When the genetically modified pigs were just born, wipe and disinfect the ears with alcohol, select the part with fewer blood vessels on the ear tissue, cut out a piece of pig ear tissue with surgery, and establish the genetically modified pig after pre-processing on the cell ultra-bacteria workbench. Fibroblast cell line and subculture.

[0030] The second step, oocyte preparation

[0031] The sow ovary collected from the slaughterhouse is preserved in normal temperature saline and transported back to the laboratory, and the oocytes of the pig are picked and cultured in vitro.

[0032] The third step, the construction of the reconstructed embryo

[0033...

Embodiment 2

[0042] Such as figure 1 As shown, a method for obtaining genetically modified cloned pigs through continuous cloning, the specific operation process is carried out in the following steps:

[0043] The first step, the culture of genetically modified porcine fibroblast donor cells and the establishment of cell lines

[0044] When the newborn Leptin-transgenic pigs were just born, they wiped and disinfected the ears with alcohol, selected the part with fewer blood vessels on the ear tissue, cut out a piece of pig ear tissue with surgery, and put it into PBS containing 200IU / mL Pencillin-StreptomycinSolution. Take it back to the lab immediately.

[0045] Immerse the cells in 70% alcohol for 10-30 seconds on the cell ultra-bacteria workbench, then place them in a sterile petri dish to remove the hair, wash them with sterile PBS containing 100IU / mL Pencillin-Streptomycin Solution for 5-10 times, and then shred them fully. Add PBS to rinse and transfer to a 50ml culture bottle, spr...

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Abstract

The invention relates to a method for obtaining cloned pigs with modified genes through continuous cloning and belongs to the field of animal reproductive biology.According to the method, tissue fibroblasts of obtained genetically modified pigs serve as donors, donor cells are transferred into enucleated porcine oocytes through the somatic cell nuclear transfer to build a genetically modified pig reconstructed embryos, then electro-fusion and electro-activation are conducted, the donor cells are transferred into estrous surrogate sow fallopian tube expanded portions after being cultured in a pig embryo in-vitro culture solution for 3 h, and after gestation of surrogate sows last for 114 d, a large scale of genetically modified pig individuals are obtained through delivery.The method can solve the problems that in the process that genetically modified individuals are obtained through the gene editing technology, target genetically modified individuals are low in efficiency, and difference between the target genetically modified individuals is large, so that genetically modified cloned pig individuals stable in target gene expression and phenotype can be efficiently produced on a large scale.

Description

technical field [0001] The invention relates to a method for producing genetically modified cloned pigs through continuous cloning technology, belonging to the field of reproductive biology. Background technique [0002] In recent years, the in-depth research and construction of genetically modified pigs as human disease models has not yet been comprehensive, and there are shortcomings such as low construction efficiency and narrow application range. Animal disease models established from the perspective of animal genetically modified cloned pigs are conducive to in-depth exploration of human-related diseases It has important reference value for the construction of clinical models of human diseases and clinical transformation applications, especially for the study of genetically modified cloned pigs obtained by serial cloning methods as animal disease models. The repeatability of the source, the reliability of the research and the stability of the genetics, the pathological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/877A01K67/027
CPCC12N15/8778A01K67/0273A01K2227/108A01K2267/03
Inventor 魏红江李鸿辉卿玉波赵红业角德灵
Owner 魏红江
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