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Method for improving in-vitro development efficiency of porcine cloned embryos

A high-efficiency technology for cloning embryos, applied in the field of reproduction or insemination, can solve the problems of low in vitro development efficiency of pig cloned embryos, and achieve the effects of increasing the number of cells at the blastocyst stage, high blastocyst rate, and increasing the division rate

Inactive Publication Date: 2013-10-02
SUN YAT SEN UNIV
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Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the defect of low in vitro development efficiency of pig cloned embryos in the prior art, and provide a method for improving the in vitro development efficiency of pig cloned embryos

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  • Method for improving in-vitro development efficiency of porcine cloned embryos
  • Method for improving in-vitro development efficiency of porcine cloned embryos
  • Method for improving in-vitro development efficiency of porcine cloned embryos

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Embodiment 1

[0032] 1. Materials

[0033] αMEM culture medium was purchased from Life Technologies; reagents were purchased from sigma; antibodies were purchased from eBioscience; plasmids pLv-CMV-ZsGreen-hOct4 (O, carrying human Oct4 gene sequence, Genbank: NM_002701.4), pLv-CMV-ZsGreen -hKlf4 (K, carrying human Klf4 gene sequence, Genbank: NM_004235.4), pLv-CMV-ZsGreen-hc-Myc (M, carrying human c-Myc gene sequence, Genbank: NM_002467.4), pLv-CMV-ZsGreen -hLin28 (L, carrying human Lin28 gene sequence, Genbank: NM_024674.4), pLv-CMV-ZsGreen-hNanog (N, carrying human Nanog gene sequence, Genbank: NM_024865.2), pLv-CMV-ZsGreen-hSox2 (S , carrying the human Sox2 gene sequence, Genbank: NM_003106.3) was preserved by the Animal Genetic Engineering Laboratory of the School of Life Sciences, Sun Yat-sen University.

[0034] Two, test procedure: the whole flow diagram of the present invention sees as figure 1 shown.

[0035] S1. Isolation and purification of bone marrow mesench...

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Abstract

The invention discloses a method for improving in-vitro development efficiency of porcine cloned embryos. The method comprises the following steps of: performing molecular identification on bone marrow-derived mesenchymal stem cells, and establishing a method capable of identifying the purity of the cells by using a few cells through a 6-channel flow type technology; performing gene modification on the porcine bone marrow mesenchymal stem cells to obtain porcine bone marrow mesenchymal stem cells for expressing multiple reprogramming factors; and separating out positive porcine bone marrow mesenchymal stem cells by using a fluorescent flow type separation method, continuously culturing the positive porcine bone marrow mesenchymal stem cells for 3 to 7 days, performing nuclear transfer on the cultured cells serving as nuclear donor cells, and studying the influence of 4RFs-3days, 6RFs-3days, 4RFs-7days and 6RFs-7days pMSC serving as nuclear donor cells on the improvement of the development efficiency of the porcine cloned embryos. The results show that the 4RFs-7days pMSC can well promote fission of the cloned embryos and formation of blastospheres, the cloned embryos can form a homogeneous state with uniform cell quantity, and a foundation is laid for efficiently cloning adult high-quality pig varieties in large scale.

Description

technical field [0001] The invention relates to a method for improving the in vitro development efficiency of pig cloned embryos, in particular to a method for improving the in vitro development efficiency of pig cloned embryos by using reprogramming factors to genetically modify donor cells, and belongs to the field of reproduction or fertilization methods. Background technique [0002] Pigs have very similar organs to humans, as well as similar anatomical and physiological characteristics, and have gradually become biological model materials for regenerative medicine research. People are increasingly using nuclear transfer technology to produce pigs with certain genetic defects or modified genes as disease models to better simulate the clinical treatment of human diseases. In addition, in the actual production of pigs, some high-quality germplasm resources cannot give full play to the breeding advantages due to the limited number of years of use, and excellent individuals ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/873
Inventor 宋振威丛佩清冀倩倩赵海静何祖勇陈瑶生
Owner SUN YAT SEN UNIV
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