Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation.
Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation.
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DOI：
10.1002/mrd.22014
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发表时间：
2012-04
影响因子：
2.5
通讯作者：
Dobrinski, I.
中科院分区：
文献类型：
--
作者：
Zeng, W.;Tang, L.;Bondareva, A.;Luo, J.;Megee, S. O.;Modelski, M.;Blash, S.;Melican, D. T.;Destrempes, M. M.;Overton, S. A.;Gavin, W. G.;Ayres, S.;Echelard, Y.;Dobrinski, I.
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 8-9 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 1.5×108 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 4 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.
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影响因子：
2.5
作者：
Honaramooz, A;Behboodi, E;Dobrinski, I
通讯作者：
Dobrinski, I
影响因子：
3.5
作者：
Nagano, M;Watson, DJ;Brinster, RL
通讯作者：
Brinster, RL
影响因子：
3.5
作者：
Nagano, M;Shinohara, T;Brinster, RL
通讯作者：
Brinster, RL
影响因子：
--
作者：
Honaramooz, A;Behboodi, E;Dobrinski, I
通讯作者：
Dobrinski, I
影响因子：
3.6
作者：
Kanatsu-Shinohara, M;Miki, H;Shinohara, T
通讯作者：
Shinohara, T