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Method for improving pig cloning efficiency on basis of inhibition of H3K9me3 methylation

A methylation, efficient technology, applied in the field of genetic engineering

Inactive Publication Date: 2016-05-04
SOUTH CHINA AGRI UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no relevant report on pig research, and there is no report showing that suppressing or erasing H3K9me3 by suppressing pig Suv39H1 and Suv39H2 genes and injecting mRNA of KDM4A, KDM4B, and KDM4D genes can improve pig cloning efficiency

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  • Method for improving pig cloning efficiency on basis of inhibition of H3K9me3 methylation
  • Method for improving pig cloning efficiency on basis of inhibition of H3K9me3 methylation
  • Method for improving pig cloning efficiency on basis of inhibition of H3K9me3 methylation

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Embodiment Construction

[0024] The present invention will be described in further detail below in conjunction with specific embodiments.

[0025] The KDM4A gene sequence involved in the present invention is derived from GenBankaccessionnumber: NM_014663.2; the KDM4B gene sequence is derived from GenBankaccessionnumber: NM_172132.2; the KDM4D gene sequence is derived from GenBankaccessionnumber: NM_173433.2; the Suv39H1 gene sequence is derived from GenBankaccessionnumber: NM_003173.3; Suv39 The gene sequence comes from GenBankaccessionnumber: NM_001039747.1.

[0026] 1. Construct KDM4A, KDM4B, KDM4D in vitro expression plasmids and obtain in vitro transcribed mRNA.

[0027] According to the KDM4A, KDM4B and KDM4D gene sequences provided by GenBank, use the plasmid analysis software to find the appropriate restriction sites, construct the coding region sequences of the three gene sequences into the pcDNA3.1+ dual-core expression vector, and artificially synthesize KDM4A and KDM4B , KDM4D gene expressio...

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Abstract

The invention discloses a method for improving the pig cloning efficiency on the basis of inhibition of H3K9me3 methylation. According to the method, H3K9me3 methylation is inhibited by inhibiting expression of Suv39H1 and Suv39H2 genes in nuclear donor cells during nuclear transplantation of a cloned embryo of a pig and overexpression of KDM4A mRNA during nuclear transplantation of a reconstructed embryo, and the pig cloning efficiency is further improved. Specifically, according to the method for improving the pig cloning efficiency, effective siRNA resisting Suv39H1 and Suv39H2 genes is co-transfected in the nuclear donor cells before nuclear transplantation, overexpression of the Suv39H1 and Suv39H2 genes in the nuclear donor cells is inhibited, accordingly, the high methylation level of H3K9me3 in the nuclear donor cells is inhibited, the processed nuclear donor cells are subjected to nuclear transplantation, cytoplasm microinjection of KDM4A mRNA is performed after the reconstructed embryo obtained after nuclear transplantation is activated, methylation of histone H3K9me3 in the reconstructed embryo is further inhibited, and the cloning efficiency of the pig is finally improved.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to transgenic engineering technology, in particular to a method for improving pig cloning efficiency. Background technique [0002] With the mass production of cloned animals, the basic method of cloning tends to be finalized and standardized, but the production efficiency of cloned animals is still very low, and the average birth rate of offspring does not exceed 5%. The low cloning efficiency makes the production of cloned animals have been in the dilemma of high input and low output, which has brought a series of problems for research and application. Pig cloning technology has been established for more than 14 years, and a variety of cloned pigs or genetically modified cloned pigs have been produced, but the efficiency of cloned pigs is still very low, and the efficiency of transplanted pig cloned embryos from developing to birth in the uterus of surrogate sows is av...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/89A01K67/027
CPCA01K67/0273A01K2207/05A01K2227/108C12N15/113C12N15/8509C12N15/8778C12N15/89
Inventor 吴珍芳李紫聪李果刘德武蔡更元郑恩琴
Owner SOUTH CHINA AGRI UNIV
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