The Study On Transgenic Chickens Mediated By Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs), as the originated cells of germs in male, which was immortalized and could be proliferate in the whole developmental process. Up to date, SSCs is a focus in the field of developmental biology of animal spermatozoa, which has widely prospects of application in clinical of veterinarian and producing transgenic animals. In this study, SSCs were transfected by exogenous DNA (pEGFP-N1) in vivo and vitro, and detected the expression of pEGFP-N1 in contemporary era. Then in F1 and F2, we detected the expression of pEGFP-N1 in sperm, embryo discs and hatching embryos in different periods, tissues, which provide the basement for producing transgenic chickens. The results were showed as following:1 .By injecting pEGFP-N1 into spermary of cock, we observed the integration of EGFP in spermatogonial stem cells and expression of offspring. Injecting pEGFP-N1 into cock and going through a spermatogenic cycle (about 25 days), we detected the transfection efficiency of spermatozoa in different time of transfection (transfection from 25 days to 240 days, detected one time by every week), and carried on southern blot detection. The result showed that after 70 days fluorescent light transfection rate of spermatozoa was highest (19.1%), and retained some time, after 180 days the rate was about 15%, after this, retained this transfection level; detected the fluorescence transfection efficiency of spermary cells of studied chicken after transfection 48 hours in 0 generation ,primordial germ cells and fibroblast of 10 days embryonic age. The result showed that spermary cells of after transfection 48 hours had EGFP, the fluorescent light transfection rate of primordial germ cells and fibroblast were 50.00%and 66.67%respectively. And then we gathered spermary tissue in 0 generation and singulorum tissues (embryonic disc,embryon,heart,kidney,liver and muscle) in offspring, made these tissues into frozen section and carried on southern blot detection, the result showed that the transfection cells surrounded to become rings in seminiferous tubules in spermary tissue in 0 generation. In chicken embryonic tissue of F1 and F2, expression of EGFP was significant difference, there was a lot of fluorescent light in kidney and liver, but there was a little in other tissues. The positive rate of embryonic disc which didn't hatch in F1and F2 were 71.69%and 67.29% respectively.The result of PCR indicated that 60.08% embryon and 69.64% embryonic disk in F1, 57.35% embryon,61.33% embryonic disk in F2 were positive. By southern blot detection, we confirmed that exogenous gene (EGFP) had been conformed into spermatozoa genome, and by fertilization in vitro the exogenous gene might express stably in descendant. So we thought that transfection pEGFP-N1 into spermatogonial stem cells in vivo was a simple and feasible way to produce transgenic chickens.2 . The spermary of the test recipient chickens that accepted SSCs transplant were dealt with Busulfan to eliminate endogenous SSCs, the result showed that the test chickens, dealt with Busulfan for 20 days, eliminate endogenous SSCs thoroughly,might keep the study of exogenous SSCs transplant in vitro. Spermatogonial stem cells taking along EGFP by electroporation, after transfection and culturing 2 days the transfection efficiency of SSCs had maxlmum, was 19.70%, and after 10 days the rate was 4.72%. Sieved the SSCs that expressed EGFP after transfection, and then the SSCs were injected into the spermary of recipient chickens. After a spermatogenic cycle (about 25 days), collected sperma in different time and detected by routine method. The result showed that after transplanting 25 days, we collected the sperma and found the sperma little became water, and the density and the transfection efficiency of spermatozoa were 1.57×104?ml-1 and 4.68% respectively. After transplanting 75 days, the sperma became light white, the density and the transfection efficiency of spermatozoa were 1.35?ml-1 and 8.02% respectively. In addition, by PCR gel electrophoresis detection of spermatozoa DNA, we observed the result was positive. After transplanting 75 days, we made testicle tissue of the test recipient chickens into frozen section, we observed that spermatogenic cells that took along exogenous gene expressed EGFP in seminiferous tubules. But the density of spermatozoa was low, we didn't satisfy the requisition of artificial insemination, so we thought that SSCs transplanting need further study to produce transgenic poultry .