| Spermatogonial stem cells (SSCs), as multipotential diploid cells which were exclusively self-renewal in animal testicular convoluted seminiferous tubule, were only stem cells which could transmit genetic information to offspring and could be isolated, purificated, cultured, cryopreservated and transplanted between homobodies or heterografts. In present study, we compared the transfection efficiencies of three different methods with SSCs from chicken embryo as experimental material, and EGFP as report gene, and emphasized the optimization of electroporation parameters. We also focused on the proper concentration of G418 in fast selection through cell toxicity sensitivity experiments, used to fast select the transfected SSCs in vitro.Then, by transplanting selected and unselected transfected cells into experimental chickens which were dealt with busulfan to eliminate endogenous SSCs, transgenic chickens were produced and they expressed EGFP protein in spermatogonial cells and semens. The results were showed as following:1. The SSCs at second generation were transfected by calcium phosphate coprecipitation method, liposome-mediated method and electroporation method with EGFP as report gene. Efficiencies of different methods were different. The highest was 19.70% by electroporation method, then it was 9.73% by liposome-mediated method, and the lowest was 2.92% by calcium phosphate coprecipitation method.2. Using EGFP as report gene, SSCs at the second passage were transfected by electroporation in order to optimize conditional parameters combination including voltage, pulse duration, plasmid concentration, temperature, cell density and cell growth stage. The results were as following: (1) under the same conditions of pulse duration, plasmid concentration, cell density, cell growth stage and temperature, the transfection rates were 13.86%,16.80%,20.09%,19.72% and 14.71% at different voltages of 170 V,220 V,270 V,320 V and 370 V , respectively. The differences between the highest transfection rate at the voltage of 270V more significantly (p<0.01) than other electric strengths except for 320 V; (2) under the same conditions of voltage, plasmid concentration, cell density, cell growth stage and temperature, the transfection rates were 13.94%,19.22%,20.27%, 13.44% and 10.30% at pulse duration of 40μs, 60μs, 80μs, 100μs and 120μs, respectively. The highest transfection rate was at 80μs more significantly than other durations except for 60μs (p<0.01); (3) under the same conditions of pulse duration, voltage, cell density, cell growth stage and temperature, the transfection rates were 9.35%, 13.82%, 20.32% and 18.76% at concentration of 5μg/mL, 10μg/mL, 15μg/mL and 20μg/mL, respectively. The transfection rate with 15μg/mL plasmid contration was highest and higher than the 5μg/mL, 10μg/mL experimental groups(p<0.01); (4) under the same conditions of pulse duration, voltage, plasmid concentration, cell growth stage and cell density, the transfection rates were 21.81%,21.70% and 10.13% at temperature of 4℃, 25℃and 37℃, respectively. However, the efficiencies at 4℃and 25℃were higher than at 37℃(p<0.05); (5) under the same conditions of pulse duration, voltage, plasmid concentration, cell growth stage and temperature, the transfection rates were 0.91%,2.64%,22.50% and 22.08% at cell density of 1×104 /ml,1×105 /ml,1×106 /ml and 1×107 /ml. The result showed that the optimum transfection density was 1×106 /mL～1×107 /mL; (6) under the same conditions of pulse duration, voltage, plasmid concentration, temperature and cell density, the transfection rate at logarithmic phase was twice than the rate at the platform phase and the difference between the efficiencies of the two groups was extremely significant (p<0.01).3. Cell toxicity sensitivity experiment was carried out to determine the tolerance of SSCs to G418 and the optimum concentration of G418 for fast selection. The SSCs could survive for 17 d,16 d,12 d,8 d,6 d,4 d,2 d,0 d,0 d and 0 d at G418 concentration of 100μg/mL,200μg/mL,300μg/mL,400μg/mL,500μg/mL,600μg/mL,700μg/mL,800μg/mL,900μg/mL and 1 000μg/mL, respectively. The results of fast selection for SSCs after transfection for 48 h at the concentration of 400μg/mL G418 medium indicated that positive SSCs clones did not appear until the sixth day, and all of the negative cells died until the eighth day.4. Experimental chickens were intraperitoneally injected with busulfan at the concentration gradient of 20 mg/Kg,25 mg/Kg,30 mg/Kg,35 mg/Kg,40 mg/Kg and 45 mg/Kg, respectively. Twenty days later, the testiculus of experimental chickens were removed and frozen sections were made. The results showed that there were no remarkable changes in the internal structures of chicken testiculus which were treated with 20 mg/Kg and 25 mg/Kg busulfan and structures of testicular eminiferous tubules in chickens which were dealt with 30 mg/Kg busulfan were impaired. The sertoli cells and spermatogenic cells decreased obviously but didn't eliminated completely; There were almost no spermatogenic cells in the chicken testiculus with the treatment of 35 mg/Kg busulfan, and the testiculus became reticulate. However, interstitial tissue structures were not damaged obviously; No spermatogenic cells were in the chicken testiculus which were dealt with 40 mg/Kg busulfan, and the seminiferous tubules became reticulate, but interstitial tissue structures were partly damaged. The seminiferous tubules of experimental chickens treated with 45 mg/Kg busulfan were reticulate and interstitial tissue structures were totally damaged. These showed that 35 mg/Kg was the optimal treated concentration of busulfan.5. The recipient chickens dealt with busulfan to eliminate endogenous SSCs were transferred with SSCs selected for 8 d and unselected. The chickens transplanted with selected SSCs were named group 1 and chickens transplanted with unselected cells were in group 2. Twenty-five days later, we collected the sperma of the two groups and found the sperma of group 1 was light ivory and generally viscous. The density of spermatozoa were 3.87(10~7 /ml) and the transfection efficiency was 4.25%. After 85 d, the density and the transfection efficiency of spermatozoa were 3.27(10~8 /ml) and 16.25%, respectively. The colour of sperma of group 2 was lighter than that of group 1, and the viscidity was lower. The density and the transfection efficiency of spermatozoa after transplanting 25 d were 1.52(10~5 /ml) and 2.68%, respectively. After 85 d, the density and the transfection efficiency of spermatozoa were 2.11(10~6 /ml) and 10.71%, respectively.6. After 25 d since the date of transplantation, genomic DNAs were extracted from the collected sperma of all experimental chickens and PCR were carried out to amplify the EGFP gene. And in the all samples, the same fragment with expected length was dected. However, the straps of amplified in group 1 were brighter than those of the group 2. We made frozen sections of testiculus tissue of the test recipient chickens and observed that spermatogenic cells that expressed EGFP existed in seminiferous tubules. But both of the fluorescent density and the fluorescent brightness in group 2 were lower than in group 1.
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