RNA Interference As a Method of Gene Knockdown in Cultured Spermatogonia

Maintenance and self-renewal of the spermatogonial stem cell (SSC) population in the testis are dictated by the expression of a unique suite of genes. In manipulating gene expression through loss-of-function approaches, we can identify important regulatory mechanisms that dictate spermatogonial fate decisions. One such approach is RNA interference (RNAi), which uses natural cellular responses to small interfering RNAs to decrease levels of a targeted transcript. RNAi is performed in primary cultures of undifferentiated spermatogonia, and can be paired with techniques such as spermatogonial transplantation to assess the functional consequences of downregulated expression of the target gene on stem cell maintenance. This approach provides an alternative or complementary strategy to the generation of knockout mouse lines / cell lines. Here, we describe the methodology of RNAi in undifferentiated spermatogonia, and outline its inherent advantages and disadvantages over other technologies in the study of gene regulation in these cells.