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A method for establishing a dox-regulated porcine somatic cell induced reprogramming system

A reprogramming and somatic cell technology, applied in the field of reprogramming of somatic cell lines, can solve the problem of unequal copy number of random insertion of four factors

Inactive Publication Date: 2016-04-27
NORTHWEST A & F UNIV
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Problems solved by technology

[0005] In order to solve the above-mentioned technical problems in the background technology, the present invention provides a method for establishing a DOX-regulated porcine somatic cell induced reprogramming system, which is convenient for studying the porcine somatic cell reprogramming system. TetO-FUW-OSKM is a four-factor four-tandem , to solve the problem of different copy numbers of four-factor random insertion, researchers can use second-generation fibroblasts to directly induce cell reprogramming under DOX conditions, without reintroducing foreign genes, without considering the efficiency of virus infection

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  • A method for establishing a dox-regulated porcine somatic cell induced reprogramming system

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Embodiment Construction

[0046] The invention provides a method for establishing a DOX-regulated porcine somatic cell induced reprogramming system, see figure 1 , the method includes the following steps:

[0047] 1. Separation steps of pig fetal fibroblasts:

[0048] The 28-day pregnant sow was taken from the pig farm as the material, and the whole uterus of the sow was brought back to the laboratory as soon as possible. Cut the uterus with surgical scissors, and place the fetuses in turn in a dish containing 1% penicillin and streptomycin in PBS (—) with forceps. In the ultra-clean bench, use two tweezers to separate the amniotic membrane and placenta from the fetal pig, put the fetal pig into new PBS (—) containing double antibodies, and wash it several times to remove the blood. Remove the head, tail, limbs and viscera of the washed fetal pig, that is, only the trunk is taken, and the blood on the tissue is removed. Add a small amount of PBS (—) to a new petri dish, cut the tissue block as much ...

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Abstract

The invention discloses a method for establishing a DOX-regulated porcine somatic cell induced reprogramming system. The method comprises the steps of 1) performing primary separation and culture on porcine embryonic fibroblasts; 2) reprogramming the porcine embryonic fibroblasts to be first-generation iPS (induced pluripotent stem); 3) differentiating the first-generation iPS to be porcine second-generation fibroblasts; and 4) reprogramming the second-generation fibroblasts to be second-generation iPS. By adopting the method disclosed by the invention, the DOX-induced porcine somatic cell reprogramming system is successfully established; porcine first-generation iPS of which the copy numbers of four factors are consistent is successfully established; the second-generation fibroblasts with consistent gene types are obtained; the second-generation fibroblasts can be successfully induced to form second-generation iPS directly under the DOX condition without gene introduction; the reprogramming process of the second-generation fibroblasts does not need re-introduction of foreign genes, and does not need to consider the virus production and infection efficiency, thus improving the safety of experiments. The method disclosed by the invention provides new resources for researching porcine reprogramming mechanisms and induced reprogramming of small molecular compounds.

Description

technical field [0001] The invention relates to a method for establishing reprogramming of somatic cell lines, in particular to a method for establishing a system for inducing reprogramming of pig somatic cells regulated by DOX. Background technique [0002] Pluripotent stem cells have the ability of self-renewal and differentiation, which has far-reaching significance for cell therapy. Embryonic stem cells (Embryonic stem cells, ESCs) are pluripotent stem cells isolated from the inner cell mass (ICM), which have self-renewal ability and three-germ layer differentiation ability, but their application is restricted due to ethical issues involving embryos. Adult stem cells can also be used in cell therapy, but their limited differentiation ability greatly limits the application of adult stem cells. The emergence of induced pluripotent stem cells has solved the above problems well. [0003] After more than ten years of hard work, pluripotent stem cell biology has become a thr...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
Inventor 王华岩段安琴于童王宁
Owner NORTHWEST A & F UNIV
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