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Method for inducing bovine induced pluripotent stem cells

A pluripotent stem cell and inducible technology, applied in the field of cell mutagenesis, can solve the problems that bovine induced pluripotent stem cells have not been reported in the literature, and achieve a stable growth state

Inactive Publication Date: 2011-11-02
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, there is no literature report on the induction technology of bovine induced pluripotent stem cells at home and abroad.

Method used

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

Examples

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specific Embodiment

[0055] Specific example: induction of Holstein cow fetal fibroblasts into induced pluripotent stem cells method:

[0056] (1), construction of defined factor lentiviral expression vector:

[0057] According to the mRNA sequences of porcine Sox2 (NM 001123197), c-Myc (NM 001005154), 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'

[0062] Upstream of pc-Myc: 5'-GCCAAGCTTGCCACCCTGGATTTCCTTCGGATAG-3'

[0063] Downstream: 5'-AAAGGATCCGCTGGGCAAGAGTTCCGTAGCTG-3'

[0064] Upstream of hOct4: 5'-AATGTCGACGCCACCATGGCGGGACACCTGGCTTC-3'

[0065] Downstream: 5'-CGCGGATCCGCTCAGTTTGAATGCATGGGAGAGC-3'

[0066] Vector m...

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Abstract

The invention discloses a method for inducing bovine induced pluripotent stem cells, which comprises: constructing a fusion protein lentivirus expression vector for inducing bovine induced pluripotent stem cells by using an exogenous defined factor and enhanced green fluorescent protein (EGPF) reporter gene; cultivating and passing bovine fetus fibroblast cells in which the fusion protein of the exogenous defined factor is expressed; gradually separating and cultivating cell colonies with clearly defined colony borders; and obtaining the bovine induced pluripotent stem cells, if the growth states of the cell colonies are stable, the karyotype is normal, the cell colonies are negative in alkaline phosphatase test, the expression of proteins Oct4, Nanog and SSEA-1 is positive in immunocyte chemical tests, teratoma having three embryonic layers can be differentiated in vivo, and the result proves the separated cell conolnies have the characteristics of embryonic stem cells.

Description

technical field [0001] The invention relates to the technical field of cell mutagenesis, in particular to a method for inducing bovine induced pluripotent stem cells. Background technique [0002] Since 2006 (Takahashi K and Yamanaka S, 2006) it was first reported that mouse skin fibroblasts were reprogrammed into induced pluripotency by exogenously introducing four defined factors (Oct4, Sox2, c-Myc, Klf4) After induced pluripotent stem cells (iPS), so far, induced pluripotent stem cells have been successfully induced from various types of somatic cells including humans, monkeys, rats and pigs (Yu J et al ., 2007; Liu H et al., 2008; Liao J et al., 2009; Esteban MA et al., 2009). Also, as research has progressed, approaches have been attempted to address issues of low efficiency and safety in the initial induction of mouse and human iPS cells. And recently, two research groups in my country reported that the mouse induced pluripotent stem cells they induced had obtained l...

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 曹鸿国章孝荣张运海李运生殷慧群陶勇刘亚方富贵刘旭光任春环张子军丁建平刘洪瑜王力生
Owner ANHUI AGRICULTURAL UNIVERSITY
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