Abstract: Objective To set up a feasible method of differentiation of male germ cells from mouse nuclear transfer embryonic stem cell in vitro. Methods We constructed mouse reconstructed embryos. The NT-ESCs like colonies were identified. The NT-ESCs were digested and divided into 4 groups : group ⑴ were induced to form embryoid bodies and cultured in 2μmol/L retinoic acid( RA) inducing them to proliferate and differentiate into male germ cells; Then passage 1 NT-ESCs as group ⑵; Group ⑶ underwent EB culture; Group ⑷ underwent EB culture and were primary cultured. And the mouse embryonic fibroblast cell as group ⑸. Differences in mRNA expression (Oct-4、VASA、Scp3、Sry、Tex14 and β-actin) were detected by RT-PCR. The proteins of VASA and Scp3 were analyzed by immunofluorescence assay. Results Most of NT-ESCs had typical characteristics of mouse embryonic stem cells . The genes related to male germ cells development were expressed in the NT-EBs cells, and the mRNA expression of Oct-4、VASA、Scp3、Sry、Tex14 and β-actin in the induced NT-EBs were positive. Those in control groups were negative. The fluorescence intensity of Scp3 in the induced NT-EBs was high(+);The fluorescence intensity of VASA in the induced NT-EBs was higher(+) than that in the control group(±). Conclusion NT-ESCs could be differentiated into male germ cells in vitro under induction of retinoic acid.

Key words: nuclear transfer, Embryonic stem cells, male germ cells, differentiation