Priority Research Centre for Reproductive Science, Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW, Australia.
Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW, Australia.
Methods Mol Biol. 2023;2656:161-177. doi: 10.1007/978-1-0716-3139-3_9.
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.
精子发生干细胞（SSC）在睾丸中的自我更新和维持是由一套独特的基因表达所决定的。通过基因敲除的方法来操纵基因表达，我们可以确定决定精原细胞命运决定的重要调控机制。其中一种方法是 RNA 干扰（RNAi），它利用细胞对小干扰 RNA 的自然反应来降低靶向转录本的水平。RNAi 是在未分化的精原细胞的原代培养物中进行的，并且可以与精原细胞移植等技术结合使用，以评估靶基因下调表达对干细胞维持的功能后果。这种方法为敲除小鼠系/细胞系的产生提供了替代或补充策略。在这里，我们描述了未分化精原细胞中 RNAi 的方法，并概述了其相对于其他技术在这些细胞中基因调控研究中的固有优势和劣势。
