Culture and genetic modification of mouse germline stem cells
Culture and genetic modification of mouse germline stem cells
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DOI：
10.1196/annals.1411.001
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发表时间：
2007-01-01
期刊：
影响因子：
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通讯作者：
Shinohara, Takashi
中科院分区：
文献类型：
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作者：
Kanatsu-Shinohara, Mito;Shinohara, Takashi
精子发生取决于称为精原干细胞的细胞群，它们能够自我更新以支持男性一生的繁殖。 2003年，小鼠精原干细胞的长期培养被证明是成功的。在神经胶质细胞源性神经营养因子存在的情况下，从出生后的小鼠睾丸中建立生殖干（GS）细胞。这些细胞在两年内增殖（> 10（85）倍），并在移植到生精小管后使先天不育的受体小鼠恢复了生育能力。与其他经常在体外获得遗传和表观遗传变化的生殖系细胞不同，GS细胞在两年的实验期间保留了其整倍体核型和雄激素遗传印记，并且产生了正常的可育后代。使用基因捕获和基因靶向载体对 GS 细胞进行诱变，成功产生纯合敲除后代，从而为种系修饰提供了一种新方法。在基因打靶实验过程中，从出生后的小鼠睾丸中成功建立了胚胎干（ES）样细胞[即多能生殖干（mGS）细胞]。除了基因组印记模式外，这些 mGS 细胞在表型上与 ES/胚胎生殖细胞相似。它们在用于诱导ES细胞分化的条件下在体外分化成各种类型的体细胞，并且mGS细胞在注射到囊胚中时形成种系嵌合体。这些新的精原干细胞系将有助于研究精子发生机制，并且对开发新的转基因或医疗技术具有重要意义。
Spermatogenesis depends on a population of cells called spermatogonial stem cells, which self-renew to support male reproduction throughout life. In 2003, the long-term culture of spermatogonial stem cells of mice proved to be successful. In the presence of glial cell-line-derived neurotrophic factor, germline stem (GS) cells were established from postnatal mouse testis. These cells proliferated over a 2-year period (> 10(85)-fold) and restored fertility to congenitally infertile recipient mice following transplantation into the seminiferous tubules. Unlike other germline cells that often acquire genetic and epigenetic changes in vitro, the GS cells retained their euploid karyotype and androgenetic imprint during the 2-year experimental period, and they produced normal fertile offspring. Mutagenization of the GS cells was successful using gene trapping and gene targeting vectors to produce homozygous knockout offspring, thereby providing a new approach to germline modification. In the course of the gene targeting experiments, establishment of embryonic-stem (ES)-like cells was also successful [i.e., multipotent germline stem (mGS) cells] from postnatal mouse testis. These mGS cells were phenotypically similar to the ES/embryonic germ cells, except for their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under the conditions used to induce the differentiation of the ES cells, and the mGS cells formed germline chimeras when injected into blastocysts. These new spermatogonial stem-cell lines will be useful for studying the mechanism of spermatogenesis, and they have important implications for developing new transgenic or medical technologies.