过表达JHDM2A对猪iPSCs诱导效率的影响

doi:10.16431/j.cnki.1671-7236.2021.12.009

Abstract

Abstract: The purpose of this study was to investigate the effect of histone demethylase (JHDM2A) on porcine fibroblast induced pluripotent stem cell efficiency, and to explore its intrinsic molecular mechanism.Porcine fibroblasts were used as materials, doxycycline (DOX)-inducible lentiviral vectors were used to produce iPSCs, and JHDM2A was overexpressed on this basis.The effect of JHDM2A on induction efficiency was detected by alkaline phosphatase (AP) staining and induction time axis.The pluripotency of the iPSCs was determined by PCR.The protein expression of the pluripotent factors was detected by immunofluorescence.The effects of JHDM2A on the expression of pluripotent factors and histone methylation related genes after clonies formation and during clonies differentiation were detected by Real-time quantitative PCR.The results showed that the cells of the JHDM2A overexpressed group deformed on the 3rd day and formed iPSCs clonies on the 8th day, which were 1 and 2 d earlier than those of control group.The results of AP staining showed that compared with the control group, the morphology of iPSCs in the JHDM2A overexpressed group was significantly improved, the AP staining was deeper, and the induction efficiency was increased by 8 times.Conventional PCR results showed that endogenous Oct4, Sox2, Klf4, c-Myc and Nanog were expressed in JHDM2A overexpressed group, control group and porcine fibroblasts (PFF), and the expression of Oct4 in iPSCs was higher than that in PFF.Immunofluorescence results showed that Oct4, Sox2, Stat3 and JHDM2A were expressed in iPSCs of JHDM2A overexpressed group, while SSEA1 and SSEA4 were weakly expressed.Real-time quantitative PCR results showed that compared with control group and PFF, the expressions of iPSCs genes Sox2, Klf4, c-Myc, Nanog, Oct4 and Tcl1 in JHDM2A overexpressed group were significantly increased (P<0.05), and the expressions of Tfcp2l1 and Zfp57 were significantly decreased (P<0.05).After DOX was removed from P5 iPSCs culture medium in the JHDM2A overexpressed group, with the increase of generation, the expressions of Sox2 and Nanog gradually decreased, the expressions of Klf4 and c-Myc first increased and then decreased, and the expressions of Oct4, Tcl1, Tfcp2l1 and Zfp57 first decreased and then increased.These results suggested that overexpression of JHDM2A could improve the induction efficiency of porcine fibroblasts by promoting histone demethylation.

Key words: pigs; JHDM2A; iPSCs; induction efficiency

CLC Number:

S828

REN Xuan, SONG Yanxia, SUN Le, HUANG Shihai, SHI Deshun, LI Xiangping. Effect of Overexpression of JHDM2A on iPSCs Induction Efficiency in Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4412-4421.

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Effect of Overexpression of JHDM2A on iPSCs Induction Efficiency in Pigs

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