Culture method of transgenic swine, and applications thereof

Abstract

The invention discloses a culture method of transgenic swine. The culture method at least comprises following steps: swine primary somatic cells are introduced into messenger RNA coded cell reprogramming genes and / or miR302 / 367 clusters for inductive production of RNA inductive multipotential stem cells; under the effect of Cas9-gRNA and CRE homologous recombinase, swine toxic genes are knock out,and knock of a plurality of human gene segments is carried out, human derived vWF gene is used for replacing swine endogenous vWF gene, and obtaining PERV-virus-inactivated human-immunological-rejection-free coagulation-incompatibility-free genes; clean target cell lines without off-target-effect are selected, and are culture into swine somatic cells; somatic cell nucleus transfer technology is adopted to treat swine somatic cells so as to obtain transgenic swine. The transgenic swine possesses following advantages, permanent PERV virus expression is inhibited permanently, human immunologicalrejection is avoided, and blood coagulation compatibility is achieved, and can be taken as human heterogeneous organ, tissue, and cell donors.

Description

technical field [0001] The invention relates to the field of transgenic technology, in particular to a breeding method and application of a transgenic pig. Background technique [0002] Organ transplantation is an effective treatment for human terminal organ failure, but it is severely limited by the shortage of donor organs, and a large number of people die each year while waiting for organs. In addition, the high cost of organ transplantation also limits the number of human organ transplant operations. In terms of the number of donor organs and the cost of organ transplantation, people think of heterologous transplantation, that is, transplanting the corresponding organs of other animals into the human body that needs organ transplantation. Among many animal organs, the size and physiological functions of pig organs are similar to those of humans, so they are recognized as the best donors for xenotransplantation. However, there is porcine endogenous retrovirus (PERV) in ...

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Problems solved by technology

[0008] The present invention aims at the existing problems of PERV virus expression, human immune rejection and coagulation incompatibility in the existing human-pig allografts, as well as the cumbersome screening process, low cloning rate, incomplete genetic modification and inability to form pig iPS in the existing solutions to the above problems Problems such as embryos, providing a breeding method for transgenic pigs

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