Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Biol Reprod. 2011 Jul;85(1):208-17. doi: 10.1095/biolreprod.111.090837. Epub 2011 Apr 6.
Spermatogonial stem cells (SSCs) are the only stem cells in the body with germline potential, which makes them an attractive target for germline modification. We previously showed the feasibility of homologous recombination in mouse SSCs and produced knockout (KO) mice by exploiting germline stem (GS) cells, i.e., cultured spermatogonia with SSC activity. In this study, we report the successful homologous recombination in rat GS cells, which can be readily established by their ability to form germ cell colonies on culture plates whose surfaces are hydrophilic and neutrally charged and thus limit somatic cell binding. We established a drug selection protocol for GS cells under hypoxic conditions. The frequency of the homologous recombination of the Ocln gene was 4.2% (2 out of 48 clones). However, these GS cell lines failed to produce offspring following xenogeneic transplantation into mouse testes and microinsemination, suggesting that long-term culture and drug selection have a negative effect on GS cells. Nevertheless, our results demonstrate the feasibility of gene targeting in rat GS cells and pave the way toward the generation of KO rats.
精原干细胞（SSCs）是体内唯一具有种系潜能的干细胞，这使得它们成为种系修饰的有吸引力的靶标。我们之前曾展示过利用生殖干细胞（GS）细胞（即具有 SSC 活性的培养精原细胞）在小鼠 SSCs 中进行同源重组的可行性，并产生了敲除（KO）小鼠。在这项研究中，我们报告了在大鼠 GS 细胞中同源重组的成功，这可以通过其在亲水和中性电荷的培养板上形成生殖细胞集落的能力来实现，从而限制了体细胞的结合。我们建立了在低氧条件下 GS 细胞的药物选择方案。Ocln 基因的同源重组频率为 4.2%（48 个克隆中有 2 个）。然而，这些 GS 细胞系在异种移植到小鼠睾丸和微受精后未能产生后代，这表明长期培养和药物选择对 GS 细胞有负面影响。尽管如此，我们的结果表明在大鼠 GS 细胞中进行基因靶向的可行性，并为产生 KO 大鼠铺平了道路。
