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

Abstract

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.

Isolation, genetic manipulation, and transplantation of canine spermatogonial stem cells: progress toward transgenesis through the male germ-line

“…In the first study a fractionated dose of 9 Gy, 8 weeks prior to transplantation was used to eliminate endogenous stem cells. While this regimen was consistent with those used successfully in other large animals (Kim et al 2006, Herrid et al 2009, Jahnukainen et al 2011, Zeng et al 2012), it may have delivered too high a dose for the dog, since testis size, sperm count and libido did not recover and sperm had to be recovered from the epididymis. Therefore, we reduced the irradiation dose to 3 Gy in the present study.…”

“…The development of SSC transplantation in rodents, as an alternative to embryo-mediated gene transfer, opened a new opportunity to explore this relatively less invasive, more efficient and lower cost approach in canines and other larger animal models. Successful SSC transplantations in several large animals (Izadyar et al 2003, Kim et al 2008, Herrid et al 2011, Jahnukainen et al 2011, Hermann et al 2012, Zeng et al 2012), led us believe this approach could be applied to the dog model.…”

“…SSC transplantation, and subsequent donor sperm production has now been reported in pigs, sheep, bulls, goats, monkeys and dogs (Izadyar et al 2003, Kim et al 2008, Herrid et al 2011, Jahnukainen et al 2011, Hermann et al 2012, Zeng et al 2012, Zeng et al 2013). More importantly, SSC transplants in both sheep (Herrid et al 2009) and goats (Honaramooz et al 2003) have led to the birth of donor-derived offspring through normal mating.…”

Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine

“…SSC transplantation is an important tool for several other biotechnological applications such as gene [75] , rats [78] , goats [81] , pigs [82] …”

“…Advances with farm animal SSCs continued in parallel. For instance, goat transgenic sperm for human growth hormone was derived from male goats that had been transplanted with SSCs previously manipulated in vitro [81] . In a report from the same group, pig embryos, transgenic for a reporter gene (enhanced green fluorescent protein), could be obtained after oocytes were fertilized with transgenic sperm from culture transduced and later transplanted SSCs [82] .…”