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Production method for buffalo induced pluripotent stem cells

A pluripotent stem cell and stem cell technology, which is applied in the field of buffalo induced pluripotent stem cell production, can solve the problems of not obtaining a buffalo embryonic stem cell line, difficulty in establishing a line, and low clone formation rate, and achieve the effect of making up for the difficulty of separation

Inactive Publication Date: 2012-05-02
GUANGXI UNIV
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Problems solved by technology

However, because the buffalo ES cells derived from the inner cell mass of the embryo are limited by materials, low clone formation rate, and difficulty in line establishment, no true buffalo embryonic stem cell line has been obtained so far.

Method used

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  • Production method for buffalo induced pluripotent stem cells
  • Production method for buffalo induced pluripotent stem cells
  • Production method for buffalo induced pluripotent stem cells

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Experimental program
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Embodiment Construction

[0027] 1. Cloning of buffalo-specific transcription factors and construction of retroviral expression vectors

[0028] According to literature reports, Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28 are expressed in the primary germ stem cells of the fetal genital ridge. The total RNA of the genital ridge was extracted by the Trizol method, and the cDNA obtained by inversion with OligodT primers and AMV reverse transcriptase (25 ° 5 min, 42 ° 1 h, 95 ° 5 min) was stored at -20 ° for later use. Specific primers were used to amplify the open reading frame (ORF) of buffalo-induced stem cell-related transcription factors. The selected primers, restriction sites, and annealing temperature are shown in Table 1. The length of each gene fragment is as follows: figure 1shown. The PCR products were recovered from the gel, connected to the pMD18-T vector, and finally sequenced to verify that these fragments were correct.

[0029] The retroviral vector used in the present invention uses pMX ...

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Abstract

The invention discloses a production method for buffalo induced pluripotent stem cells. Specific transcription factors such as Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28 and a cell immortal gene SV40large T (LT) are carried by retrovirus vectors, and buffalo somatic cells such as fetus fibroblasts are directly reprogrammed into the induced pluripotent stem cells with characteristics similar to those of embryonic stem cells. Due to the generation of the buffalo induced pluripotent stem cells, a new method for obtaining buffalo stem cells can be provided, the problems that the buffalo embryonic stem cells are difficult to separate and cultivate, and cell lines are difficult to establish are solved to some extent, moreover, sources of stem cells are provided for the research of signal paths of the buffalo stem cells and the establishment of the cell lines, and a new method is provided for directional genetic improvement of buffalos at the same time.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a method for producing buffalo induced pluripotent stem cells. Background technique [0002] In 1981, Evans et al. established an ES (embryonic stem cell) cell line derived from the inner cell mass (ICM) of mouse blastocysts. In the 1990s, Thomson et al. also obtained human and primate ES cell lines. Embryonic stem cells have two special properties, one is the ability to maintain unlimited proliferation and self-renewal; the other is the potential to differentiate into various tissue cells, that is, to differentiate into various tissues and cells including the three germ layers. In view of the special properties of human embryonic stem cells, it has broad application prospects in regenerative medicine, such as stem cell transplantation, directed differentiation and tissue regeneration, which can promote wound repair and disease treatment. Because the acquisition of human embryonic ste...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/867
Inventor 石德顺邓彦飞刘庆友罗婵
Owner GUANGXI UNIV
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