Method for inducing porcine induced pluripotent stem cells

A pluripotent stem cell and inducible technology, applied to embryonic cells, animal cells, vertebrate cells, etc., to achieve a stable growth state

Inactive Publication Date: 2011-01-26
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, none of the three research groups mentioned above used the fusion protein of defined factors to induce porcine induced pluripotent stem cells.

Method used

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  • Method for inducing porcine induced pluripotent stem cells
  • Method for inducing porcine induced pluripotent stem cells
  • Method for inducing porcine induced pluripotent stem cells

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Experimental program
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Embodiment Construction

[0054] Pig induced pluripotent stem cell induction method:

[0055] (1), construction of defined factor lentiviral expression vector

[0056] Reagents: restriction endonuclease, cDNA first-strand synthesis kit, T4 DNA polymerase and ligase were purchased from TaKaRa Biotechnology Co., Ltd.; Taq DNA polymerase and NTP were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; Primer synthesis and sequencing were completed by Shanghai Bioengineering Co., Ltd.

[0057] Method: According to the mRNA sequences of porcine Sox2 (NM 001123197), c-Myc (NM 001005154), and Klf4 (NM001031782) genes on NCBI, the upstream and downstream primers were designed and synthesized. The primer sequences are as follows:

[0058] Upstream of pSox2: 5'-TTTAAGCTTGCCACCATGTACAACATGATGGAGAC-3'

[0059] Downstream: 5'-AAAGGATCCGGCATGTGAGAGAGAGGCAGTGTAC-3'

[0060] Upstream of pKlf4: 5'-TATAAGCTTGCCACCATGGCTGTCAGCGACGCAC-3'

[0061] Downstream: 5'-GGCGGATCCGGAAAATGCCTCTTCATGTGTAAGGC-3' ...

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Abstract

The invention discloses a method for inducing porcine induced pluripotent stem cells, which comprises the following steps of: constructing a fusion protein lentivirus expression vector for inducing the porcine induced pluripotent stem cells by using an exogenous defined factor and an enhanced green fluorescent protein (EGFP) reporter gene; subculturing porcine embryo fibroblasts for expressing exogenous defined factor fusion protein; and separating and culturing cell clones with clear colony edges step by step to obtain the porcine induced pluripotent stem cells, wherein the growth state of cell colonies is stable, karyotype is normal, alkaline phosphatase test is positive, and immunocytochemical detection shows that the expression of Oct4, Nanog and SSEA-1 protein is positive, and teratomas with three embryonic layers can be formed by differentiation in vivo.

Description

technical field [0001] The invention relates to the field of cell induction, in particular to a method for inducing porcine induced pluripotent stem cells. Background technique [0002] For many years, people have been studying the reprogramming of cells and the potential totipotency of the nucleus. The birth of Dolly the sheep in 1998 marked that this research work has been advanced to the mammalian field. In 2006, Professor Yamanaka of Kyoto University in Japan published a landmark article on Cell (Takahashi K and Yamanaka S, 2006), detailing the use of four limiting factors (Oct3 / 4, Sox2, Klf4 and c -Myc) reversed the reprogramming of mouse fibroblasts to resemble pluripotent stem cells and the results, which ushered in the era of induced pluripotent stem cells (iPS). Since pluripotent stem cells (pluripotent stem cells) have the potential to form all types of cells in humans and animals and the characteristics of continuous self-renewal, they are widely used in cell rep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N15/62C12N15/867C12N7/01
Inventor 张运海曹鸿国章孝荣李运生殷慧群孙雪萍薛奕杰张卫琴黄伟玲陈涛宋锐陶勇刘亚方富贵刘旭光任春环张子军殷宗俊丁建平
Owner ANHUI AGRICULTURAL UNIVERSITY
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