Dann Christina Tenenhaus
Department of Chemistry, Indiana University, Bloomington, IN, USA.
Methods Mol Biol. 2013;927:503-18. doi: 10.1007/978-1-62703-038-0_43.
The continuous production of spermatazoa throughout the reproductive lifetime of a male depends on the maintenance of a pool of progenitor cells called spermatogonial stem cells (SSCs). SSCs represent a very small fraction of the cellular population in the testes and lack definitive molecular markers for their identification. The discovery of conditions that allow one to propagate mouse SSCs in vitro essentially indefinitely has truly facilitated studies of the molecular mechanisms regulating SSC function. While multiple conditions for culturing SSCs have now been described, here we detail a method for culturing SSCs that uses a simpler medium than the original formulation. As with numerous other primary and stem cell cultures, it is difficult to introduce DNA into cultured SSCs using standard transfection approaches. However, VSV-G pseudotyped lentivirus efficiently infects cultured SSCs with minimal toxicity. Here we present protocols for producing lentivirus and stably modifying the genome of cultured SSCs using lentiviral vectors.
雄性在整个生殖寿命期间持续产生精子，这依赖于一群被称为精原干细胞（SSCs）的祖细胞的维持。精原干细胞在睾丸细胞群体中只占很小一部分，并且缺乏用于鉴定它们的明确分子标记。能够在体外基本无限期地扩增小鼠精原干细胞的条件的发现，确实推动了对调节精原干细胞功能的分子机制的研究。虽然现在已经描述了多种培养精原干细胞的条件，但在此我们详细介绍一种培养精原干细胞的方法，该方法使用的培养基比原始配方更简单。与许多其他原代细胞和干细胞培养一样，使用标准转染方法很难将DNA导入培养的精原干细胞。然而，水泡性口炎病毒糖蛋白（VSV-G）假型慢病毒能以最小的毒性有效感染培养的精原干细胞。在此我们展示了生产慢病毒以及使用慢病毒载体稳定修饰培养的精原干细胞基因组的方案。
