Viral Transduction of Male Germline Stem Cells Results in Transgene Transmission after Germ Cell Transplantation in Pigs1

Abstract

Genetic modification of germline stem cells (GSCs) is an alternative approach to generate large transgenic animals where transgenic GSCs are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objective of the present study was to explore the application of viral vectors in delivering an enhanced green fluorescent protein (EGFP) transgene into GSCs for production of transgenic gametes through germ cell transplantation. Both adeno-associated virus (AAV)- and lentivirus (LV)-based vectors were effective in transducing pig GSCs, resulting in the production of transgenic sperm in recipient boars. Twenty-one boars treated with busulfan to deplete endogenous GSCs and nine nontreated boars received germ cell transplantation at 12 wk of age. Semen was collected from recipient boars from 5 to 7 mo posttransplantation when boars became sexually mature, and semen collection continued for as long as 5 yr for some boars. The percentage of ejaculates that were positive for the EGFP transgene ranged from 0% to 54.8% for recipients of AAV vector-transduced germ cells (n = 17) and from 0% to 25% for recipients of LV vector-transduced germ cells (n = 5). When semen from two AAV recipients was used for in vitro fertilization (IVF), 9.09% and 64.3% of embryos were transgenic. Semen collected from two LV-vector recipients produced 7.7% and 26.3% transgenic IVF embryos. Here, we not only demonstrated AAV-mediated GSC transduction in another large animal model (pigs) but also showed, to our knowledge for the first time, that LV-mediated GSC transduction resulted in transgene transmission in pigs.

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

“…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] . Very recently, bovine gonocytes were transduced through a lentivirus vector and the reporter transgene product proved the success of the procedure [83] .…”

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

Efficient nonmeiotic allele introgression in livestock using custom endonucleases

“…Pigs edited to disrupt the DAZL gene can serve as a model for studying the restoration of human fertility by germ cell transplantation or for producing genetically modified offspring by transfer of genetically modified germline stem cells, as has been demonstrated in pigs (45), goats (46), and rodents (47,48). Gene-edited alleles of APC also could provide a size-relevant model of colon cancer for preclinical evaluation of therapeutics, surgical intervention, or detection modalities.…”

Multipotent male germline stem cells (mGSCs) from neonate porcine testis

“…Previous studies demonstrated that several pluripotent/multipotent germline stem cells have been established and characterized by different research groups using various protocols, and murine and human male GSCs can be cultured for long periods and retain the capacity to differentiate into multiple cell types in the presence of serum or feeder cells in vitro. Recently, porcine, goat and bovine mGSCs were cultured and characterized, and these cells shared similar characteristics of ES cells and GSCs (Kanatsu-Shinohara et al 2008;Goel et al 2009;Kuijk et al 2009;Hua et al 2011;Zeng et al 2013;Zheng et al 2013;Park et al 2014). However, the methods available for isolating SSCs and mGSCs from porcine testicular cells have a low efficiency of cell separating, and it is difficult to establish a stable livestock mGSCs line Zheng et al 2013;Park et al 2014).…”