Production of knockout mice by gene targeting in multipotent germline stem cells.
Production of knockout mice by gene targeting in multipotent germline stem cells.
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DOI：
10.1016/j.ydbio.2007.09.029
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发表时间：
2007-12
影响因子：
2.7
通讯作者：
M. Takehashi;Mito Kanatsu-Shinohara;H. Miki;Jiyoung Lee;Y. Kazuki;K. Inoue;N. Ogonuki;S. Toyokuni;M. Oshimura;A. Ogura;T. Shinohara
中科院分区：
文献类型：
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作者：
M. Takehashi;Mito Kanatsu-Shinohara;H. Miki;Jiyoung Lee;Y. Kazuki;K. Inoue;N. Ogonuki;S. Toyokuni;M. Oshimura;A. Ogura;T. Shinohara
精原干细胞可以在体外转化为类似胚胎干细胞的多能生殖系干细胞，并通过胚泡显微注射产生生殖系嵌合体。虽然以前在MGS细胞中发现了同源重组，但在嵌合体中没有发现精子发生，这表明它们不能进行生殖系修饰。在这里，我们对嵌合动物进行了详细的分析，以确定MGS细胞在基因操作后是否保留了生殖系潜力。封闭蛋白基因缺陷的精子可以从与MGS细胞进行同源重组的嵌合动物中恢复。阻断蛋白基因敲除(KO)小鼠的表型与用ES细胞培育的KO小鼠相似，并表现出生长迟缓、胃炎和男性不育。此外，我们还发现，杂合的MGS细胞获得了两个G418抗性基因的拷贝，并通过在高浓度G418中培养而成为目标等位基因的纯合。细胞遗传学分析显示，在遗传操作过程中观察到的非整倍体MGS细胞是8号或11号染色体的三体，这是ES细胞中常见的染色体异常。因此，MGS细胞可以用来生产KO动物，这种新的生殖系操作方法可能在不同的哺乳动物物种中被证明是有用的。
Spermatogonial stem cells can convert into embryonic stem (ES) cell-like multipotent germline stem (mGS) cells in vitro and produce germline chimeras by blastocyst microinjection. Although homologous recombination was previously demonstrated in mGS cells, spermatogenesis was not found in chimeras, suggesting that they are not competent for germline modification. Here we conducted detailed analysis of chimeric animals to determine whether mGS cells retain germline potential after genetic manipulation. Spermatozoa that were deficient in the occludin gene could be recovered from animals that were chimeric with mGS cells that underwent homologous recombination. The phenotypes of the occludin knockout (KO) mice were similar to those reported for KO mice produced using ES cells, and the animals showed growth retardation, gastritis and male infertility. Furthermore, we found that heterozygous mGS cells acquire two copies of the G418-resistant genes and become homozygous for the targeted allele by culturing at high concentrations of G418. Cytogenetic analysis showed that the aneuploid mGS cells observed during genetic manipulation were trisomic for chromosome 8 or 11, which is a common chromosomal abnormality in ES cells. Thus, mGS cells can be used to produce KO animals, and this novel method of germline manipulation may prove useful in diverse mammalian species.