Enrichment of testicular gonocytes and genetic modification using lentiviral transduction in pigs.
Gonocytes are long-lived primary germ cells that reside in the center of seminiferous cords until differentiation into spermatogonia that drive spermatogenesis. In pigs, gonocytes have research value in the production of transgenic offspring through germline modification and transplantation. However, the rarity of pig gonocytes has raised the need for an efficient isolation method. Therefore, in this study we use components of extracellular matrix, laminin, fibronectin, and collagen type IV and their derivative, gelatin, to establish a negative selection system for functionally viable gonocytes in neonatal pig. We then demonstrate functional analysis with genetic modification using lentiviral transduction and successfully transplant the donor gonocytes, which colonized the seminiferous tubules of the recipient mouse. The most effective selection method was established by sequential use of laminin and gelatin, in which the purity of gonocytes was 80% and the recovery rate of gonocytes was 78%. The selected gonocytes were labeled with fluorescent dye PKH26 and transplanted into busulfan-treated immunodeficient mouse testes. The fluorescent gonocytes colonized the recipient testes, and the resultant germ cell colonies were visible up to 4 mo after transplantation. When gonocytes were transplanted after transduction with an enhanced green fluorescent protein marker gene using lentiviral vectors, the transduced germ cell colonies were visible up to 6 mo and displayed an estimated transduction efficiency of 11.1%. These results can be applied and extended to isolate and enrich gonocytes of other species for in vitro and in vivo studies and to assist in genetic modification of male germline stem cells of livestock species.
Kim
Byung-Gak
BG
Department of Animal Science and Technology, Chung-Ang University, Ansung-Si, Gyeonggi-Do, Korea.
Cho
Chul Min
CM
Lee
Yong-An
YA
Kim
Bang-Jin
BJ
Kim
Ki-Jung
KJ
Kim
Yong-Hee
YH
Min
Kwan-Sik
KS
Kim
Chul Geun
CG
Ryu
Buom-Yong
BY
D000818:Animals; D002066:Busulfan / Q000494:pharmacology; D002469:Cell Separation / Q000379:methods*; D024141:Collagen Type IV / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005780:Gelatin / Q000378:metabolism; D005822:Genetic Vectors; D005854:Germ Cells / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007797:Laminin / Q000378:metabolism; D016086:Lentivirus; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D012641:Selection, Genetic; D013552:Swine; D013737:Testis / Q000166:cytology*; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous
D024141:Collagen Type IV; D005353:Fibronectins; D007797:Laminin; D049452:Green Fluorescent Proteins; D005780:Gelatin; D002066:Busulfan