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首页> 外文期刊>Molecular reproduction and development >Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation
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Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation

机译：通过核转染非病毒转染山羊种系干细胞可导致生殖细胞移植后产生转基因精子

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Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 89 weeks post-irradiation. Donor cells were collected from testes of 9-week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5 x 10(8) cells in 3ml, and transplanted into one testis (n=4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3%+/- 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats. Mol. Reprod. Dev. 79: 255261, 2012
机译：生殖细胞干细胞（GSC）可用于大型动物转基因，其中将经过体外基因操作的GSC移植到受体睾丸中，以产生供体来源的转基因精子。这项研究的目的是探索一种非病毒的方法来将基因转染到山羊GSC中，并研究核转染在产生转基因精子中的效率。四只受体山羊在8周龄时接受了分级辐照，以耗尽内源性GSC。照射后89周进行生殖细胞移植。从9周龄山羊的睾丸中收集供体细胞，通过Staput速度沉淀法富集GSC，并用转染基因的转染子转染，该转基因构建物在山羊β-酪蛋白启动子（GBC）和启动子上游的鸡β-珠蛋白绝缘子（CBGI）序列。对于每个受体，将来自10个核转染反应的转染细胞合并，与未转染的细胞混合，混合成3ml的1.5 x 10（8）个细胞，然后通过超声引导的导管插入一个睾丸（n = 4个受体）后睾丸。每个收件人的第二个睾丸被删除。从移植后的9个月开始收集精液，历时一年以上（总共从4位接受者处抽出62粒精液）。 hGH和CBGI序列的巢式基因组PCR结果表明，射精的hGH和CBGI阳性率为31.3％+ /-12.6％。这项研究提供了概念上的证明，即原代山羊生殖细胞的非病毒转染（核转染），然后进行生殖细胞移植，会导致转基因传递给受体山羊的精子。大声笑责备。开发人员79：255261，2012年

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