Production of transgenic mice by random recombination of targeted genes in female germline stem cells
Production of transgenic mice by random recombination of targeted genes in female germline stem cells
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通过随机重组雌性生殖干细胞中的目标基因来生产转基因小鼠。
DOI：
10.1093/jmcb/mjq043
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发表时间：
2011-04-01
影响因子：
5.5
通讯作者：
Wu, Ji
中科院分区：
文献类型：
--
作者：
Zhang, Yong;Yang, Zhaojuan;Wu, Ji
大多数哺乳动物的卵母细胞生产被认为在出生前停止。然而，这一想法受到了出生后小鼠卵巢具有有丝分裂活性生殖细胞的发现的挑战。最近的一项研究表明，成年小鼠的雌性生殖干细胞（FGSCs）被分离、长期培养，并在移植到不育小鼠体内后产生卵母细胞和后代。在这里，我们证明了使用FGSC成功产生转基因或基因敲除小鼠。从5日龄和成年小鼠卵巢分离FGSCs，分别用携带绿色荧光蛋白、卵母细胞-G1或小鼠动力蛋白轴丝中间链2基因的重组病毒感染，或用卵母细胞-G1特异性shRNA表达载体（pRS shOocyte-G1 vector）转染，然后移植到不育小鼠体内。卵巢中移植的细胞进行卵子发生，并在与野生型雄性小鼠交配后产生杂合子后代。后代的遗传特征和转移或敲低基因的生物学功能进行了研究。转基因或基因敲除的效率为29%-37%，产生转基因后代需要2个月。FGSCs的基因操作是一种快速、有效的动物转基因方法，有望成为生物医学和生物技术的有力工具。
Oocyte production in most mammalian species is believed to cease before birth. However, this idea has been challenged with the finding that postnatal mouse ovaries possess mitotically active germ cells. A recent study showed that female germline stem cells (FGSCs) from adult mice were isolated, cultured long term and produced oocytes and progeny after transplantation into infertile mice. Here, we demonstrate the successful generation of transgenic or gene knock-down mice using FGSCs. The FGSCs from ovaries of 5-day-old and adult mice were isolated and either infected with recombinant viruses carrying green fluorescent protein, Oocyte-G1 or the mouse dynein axonemal intermediate chain 2 gene, or transfected with the Oocyte-G1 specific shRNA expression vector (pRS shOocyte-G1 vector), and then transplanted into infertile mice. Transplanted cells in the ovaries underwent oogenesis and produced heterozygous offspring after mating with wild-type male mice. The offspring were genetically characterized and the biological functions of the transferred or knock-down genes were investigated. Efficiency of gene-transfer or gene knock-down was 29%-37% and it took 2 months to produce transgenic offspring. Gene manipulation of FGSCs is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.