Construction method of cloned pig in which porcine endogenous retrovirus is inactivated

Abstract

The invention relates to a construction method of a cloned pig in which a porcine endogenous retrovirus is inactivated, and belongs to the field of animal biotechnology. Through a ddPCR and whole genome sequencing comparison, the copy number of the porcine endogenous retrovirus in a porcine fetal fibroblast genome and the position of the virus on a chromosome are determined, a CRISPR-Cas9 technology is utilized for constructing a porcine endogenous retrovirus inactivated vector to be transfected to porcine fetal fibroblasts, screened positive cells are subjected to somatic cell nuclear transfer, during gestation, a fetus is taken out to identify whether the virus is inactivated or not, after the ddPCR and whole genome sequencing comparison are utilized, the porcine fetal fibroblasts are subjected to gene targeting, sequential circulation is conducted until the cells in which the porcine endogenous retrovirus is completely inactivated are obtained, then somatic cell nuclear transfer isconducted, and finally, the cloned pig in which the porcine endogenous retrovirus is completely inactivated is obtained. The construction method solves the problems that the inactivation efficiency ofthe porcine endogenous retrovirus is low, and the success rate is low, and has an important significance in solving the safety problems of xenotransplantation.

Description

technical field [0001] The invention belongs to the field of animal biotechnology, in particular to a method for constructing pig endogenous retrovirus inactivated cloned pigs. Background technique [0002] Organ transplantation is the only way to treat patients with end-stage organ failure, while xenotransplantation is the most effective way to solve the severe shortage of human organ donors. In recent years, with the substantial increase of patients with chronic diseases such as diabetes, chronic kidney disease, cardiovascular disease and hepatitis in my country, the number of patients with end-stage liver, kidney, heart and other organ failure is increasing sharply, and the pressure on organ demand will also increase. getting bigger. According to incomplete statistics, the number of clinical organ transplants that can be performed in my country is only about 10,000 cases per year, while there are 1.5 million patients waiting for organ transplants. The ratio of the number ...

AI Technical Summary

Problems solved by technology

Reduce the risk of porcine PERV infection through small interfering RNA (Ramsoondar et al., 2009) and use ZNF technology (Semaan et al., 2015), TALENs technology (Dunnet al., 2015) to eliminate PERV, these methods Neither can avoid the risk of PERV infection

In 2015, Yang Luhan’s team knocked out all PERV genes in pig cells through CRISPR / Cas9 technology (Yang et al., 2015). In 2017, the world’s first PERV-inactivated cloned pigs were successfully born in Yunnan (Niu et al. , 2017), making xenotransplantation an important step towards clinical practice, but the copy numbers of endogenous retroviruses in different pig species are different, and the inactivation process of endogenous retroviruses is difficult, and gene editing Slower cell growth and less success in obtaining cell lines with complete porcine endogenous retrovirus inactivation

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