School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, No. 800 Dongchuan Road, Minhang District, Shanghai 200240, China.
J Mol Cell Biol. 2011 Apr;3(2):132-41. doi: 10.1093/jmcb/mjq043. Epub 2010 Dec 10.
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.
大多数哺乳动物物种的卵母细胞生产被认为在出生前就停止了。然而，这一观点受到了挑战，因为发现产后小鼠的卵巢具有有丝分裂活性的生殖细胞。最近的一项研究表明，从成年小鼠中分离出雌性生殖干细胞（FGSCs），经过长期培养，在移植到不孕小鼠后产生卵母细胞和后代。在这里，我们展示了使用 FGSCs 成功生成转基因或基因敲低小鼠。从 5 天大的和成年小鼠的卵巢中分离出 FGSCs，并用携带绿色荧光蛋白、Oocyte-G1 或小鼠动力蛋白轴中间链 2 基因的重组病毒感染，或用 Oocyte-G1 特异性 shRNA 表达载体（pRS shOocyte-G1 载体）转染，然后移植到不孕小鼠中。移植到卵巢中的细胞经历了卵母细胞发生，并与野生型雄性小鼠交配后产生了杂合子后代。对后代进行了遗传特征分析，并研究了转导或敲低基因的生物学功能。基因转移或基因敲低的效率为 29%-37%，产生转基因后代需要 2 个月的时间。FGSCs 的基因操作是一种快速有效的动物转基因方法，可作为生物医学科学和生物技术的有力工具。
