Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation.

  • W Zeng, L Tang, +11 authors Ina Dobrinski
  • Published 2012 in Molecular reproduction and development


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 × 10(8) cells in 3 ml, 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.

DOI: 10.1002/mrd.22014

Cite this paper

@article{Zeng2012NonviralTO, title={Non-viral transfection of goat germline stem cells by nucleofection results in production of transgenic sperm after germ cell transplantation.}, author={W Zeng and L Tang and Alla Bondareva and J Luo and Susan O Megee and Mark Modelski and Stephen Blash and David T. Melican and Margaret M. Destrempes and Susan A. Overton and William G. Gavin and S Ayres and Yann Echelard and Ina Dobrinski}, journal={Molecular reproduction and development}, year={2012}, volume={79 4}, pages={255-61} }