Establishment of paired box gene 6/mouse embryonic stem cell line and its stem cell biological characteristics

doi:10.16098/j.issn.0529-1356.2019.04.008

Abstract

Abstract:

Objective To investigate the overexpression of paired box gene 6(Pax6) gene in mouse embryonic stem cells and obtain cell line, which is the basis for further differentiation of Pax6/mouse embryonic stem cells (mESCs). Methods mESCs were cultured in vitro, and the recombinant vector pEF1α-Pax6-IRES-AcGFP and the empty vector pEF1α-IRES-AcGFP were transfected into mESCs by liposome method respectively. The cells were screened by G418 gradient and fluorescent protein. The expression of Pax6 was detected by RT-PCR, immunofluorescence and Western blotting and the proportion of Pax6/mESCs positive cells was detected by flow cytometry. The obtained cell line was detected by cell immunofluorescence for stem cell markers stage specific embryonic antigen 1(SSEA1) and octamer binding transcription factor 4(OCT4), and the pluripotency was detected by alkaline phosphatase staining. Pax6/mESCs cells were subcutaneously transplanted, and the grafts were observed by HE staining to observe their differentiation ability. Results Pax6 was successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs was obtained, and the flow rate showed positive rate about 90%. Immunofluorescence showed that stem cell markers SSEA1 and OCT4 were positively expressed and alkaline phosphatase stained cells were stained brownish black, and transplantation in vivo could differentiate into three germ layers. Conclusion Pax6 is successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs is obtained and shows the good stem cell characteristics.

Key words: Paired box gene 6, Mouse embryonic stem cell, Gene transfection, G418 screening, Flow cytometry, Mouse

DING Ling GAO Jie LI Hong HU Rong SU Min. Establishment of paired box gene 6/mouse embryonic stem cell line and its stem cell biological characteristics[J]. Acta Anatomica Sinica, 2019, 50(4): 451-458.

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