试验旨在研究梅山猪白细胞分化抗原14(cluster of differentiation antigen 14,CD14)基因多态性及其对部分免疫指标和繁殖性能的遗传效应。采用PCR-RFLP方法对梅山猪CD14基因-61bp处多态性进行分析,同时对部分重要细胞因子(IL-1β、IL-4...试验旨在研究梅山猪白细胞分化抗原14(cluster of differentiation antigen 14,CD14)基因多态性及其对部分免疫指标和繁殖性能的遗传效应。采用PCR-RFLP方法对梅山猪CD14基因-61bp处多态性进行分析,同时对部分重要细胞因子(IL-1β、IL-4、IL-6、IL-8、IL-10、IFN-γ、TNF-α和TGF-β1)水平及繁殖性能(总产仔数、断奶仔猪数、初生窝重和断奶窝重)进行测定,并分析CD14基因多态性与部分免疫指标和繁殖性能的相关性。结果显示,梅山猪CD14基因-61bp处经BamHⅠ酶切后共检测到3种基因型,分别定义为:AA、AG和GG;χ2适合性检验显示,梅山猪处于Hardy-Weinberg平衡状态(P>0.05);多态信息含量(PIC)分析显示,梅山猪群体处于中度多态。关联分析结果表明,AA和AG基因型个体的IL-10水平显著高于GG基因型个体(P<0.05);在初产母猪中,3种基因型间的繁殖性能均无显著差异(P>0.05),而在经产母猪中,AA和AG基因型个体的断奶窝重显著高于GG基因型个体(P<0.05)。本试验结果表明,基于CD14基因-61bp处多态性对梅山猪进行分子选育时,无论是一般抗病力还是繁殖性能,GG基因型可能均为不利基因型,可以将CD14基因-61bp处作为一个潜在的遗传标记进行深入系统的验证和分析。展开更多
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 te...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.展开更多
文摘试验旨在研究梅山猪白细胞分化抗原14(cluster of differentiation antigen 14,CD14)基因多态性及其对部分免疫指标和繁殖性能的遗传效应。采用PCR-RFLP方法对梅山猪CD14基因-61bp处多态性进行分析,同时对部分重要细胞因子(IL-1β、IL-4、IL-6、IL-8、IL-10、IFN-γ、TNF-α和TGF-β1)水平及繁殖性能(总产仔数、断奶仔猪数、初生窝重和断奶窝重)进行测定,并分析CD14基因多态性与部分免疫指标和繁殖性能的相关性。结果显示,梅山猪CD14基因-61bp处经BamHⅠ酶切后共检测到3种基因型,分别定义为:AA、AG和GG;χ2适合性检验显示,梅山猪处于Hardy-Weinberg平衡状态(P>0.05);多态信息含量(PIC)分析显示,梅山猪群体处于中度多态。关联分析结果表明,AA和AG基因型个体的IL-10水平显著高于GG基因型个体(P<0.05);在初产母猪中,3种基因型间的繁殖性能均无显著差异(P>0.05),而在经产母猪中,AA和AG基因型个体的断奶窝重显著高于GG基因型个体(P<0.05)。本试验结果表明,基于CD14基因-61bp处多态性对梅山猪进行分子选育时,无论是一般抗病力还是繁殖性能,GG基因型可能均为不利基因型,可以将CD14基因-61bp处作为一个潜在的遗传标记进行深入系统的验证和分析。
基金Supported by the National Natural Science Foundation of China(Grant No.30430030)Specialized Research Fund for the Doctoral Program of Higher Education(Grant No.20061117004)
文摘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.
