Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

【摘要】 The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock’s testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.更多还原

【Abstract】 The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein(GFP) gene into the cock’s testis termed as testis-medianted gene transfer(TMGT), and transplanting transfected spermatogonial stem cells(TTSSCs).For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly.The results showed:(1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively.The difference from the four dosage groups was significant(P<0.05).On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed.The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively.The difference from the four dosage groups was also significant(P<0.05).(2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization.(3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females.The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2%(254/452) and 53.2%(275/517), respectively.The detec-tion of polymerase chain reaction(PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5%(3/23) of F1 and 52.9%(9/17) of F2 were positive.Southern blot showed GFP DNA was in-serted in their genomic DNAs.(4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%.(5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted.The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes.Moreover, sperms carrying GFP could be produced normally.The results of artificially inseminating wild-type females with these sperms showed 12.5%(8/64) of offspring embryo expressed GFP and 11.1%(2/18) hatched chicks were tested transgenic.Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.更多还原