Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells.
To study self-renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA-1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty-five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (x10(7)/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (x10(7)/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples.
Yu
Fei
F
College of Animal Science and Technology, Yangzhou University, Yangzhou City, China.
Ding
Li-Jun
LJ
Sun
Guo-Bo
GB
Sun
Peng-Xiang
PX
He
Xian-Hong
XH
Ni
Li-Gang
LG
Li
Bi-Chun
BC
D000818:Animals; D030801:Animals, Genetically Modified; D053713:Azoospermia; D002470:Cell Survival; D002478:Cells, Cultured; D002645:Chickens; D053686:Fetal Stem Cells / Q000502:physiology / Q000637:transplantation*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D013093:Spermatogonia / Q000166:cytology*; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology*; D014162:Transfection / Q000379:methods*; D014184:Transplantation, Homologous
D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins