Gene Silencing of Human Sertoli Cells Utilizing Small Interfering RNAs.

ABSTRACT

Sertoli cells, as the unique somatic cells within the seminiferous tubules, play essential roles in regulating normal spermatogenesis. In addition, recent studies have demonstrated that Sertoli cells could have significant applications in regenerative medicine due to their great plasticity. However, the roles of genes in controlling the fate determinations of human Sertoli cells remain largely unknown. Silencing genes of human Sertoli cells utilizing small interfering RNAs (siRNAs) is an important method to explore their functions and mechanisms in human Sertoli cells. We isolated and identified human Sertoli cells. RNA interference (RNAi) was employed to probe the roles and signaling pathways of BMP6 and BMP4 in mediating the proliferation and apoptosis of human Sertoli cells. Specifically, siRNAs against BMP6 and BMP4 were used to knock down the expression levels of BMP6 and BMP4 and examine the function and mechanism in controlling the fate decisions of human Sertoli cells. In this chapter, we provided the detailed methods of RNAi in silencing BMP6 gene of human Sertoli cells. Quantitative real-time PCR demonstrated that the designed BMP6 siRNAs apparently silenced BMP6 mRNA in human Sertoli cells at 24 h after transfection. Western blots showed that the siRNAs silenced the expression of BMP6 protein effectively at 48 h after transfection. In summary, siRNAs can effectively and specifically knock down targeting genes at both transcriptional and translational levels utilizing RNAi in human Sertoli cells.