Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen ...Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents. Methods: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 ℃ for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light. Results: The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L -5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents. Conclusion: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.展开更多
The domesticated chicken has important roles in basic and applied research. The vector based on scaffold matrix attachment region （S/MAR） appears to be sufficient to maintain long-term expression in an episomal stat...The domesticated chicken has important roles in basic and applied research. The vector based on scaffold matrix attachment region （S/MAR） appears to be sufficient to maintain long-term expression in an episomal state in various mammalian cells. To explore the practical use of episomal vector in transgene technology of agricultural chickens, we fused the S/MAR of the human r-interferon gene into the pEGFP vector and transduced it into chickens by sperm-mediated gene transfer （SMGT）. PCR detection indicated the positive rate of transgene chickens was 60%. The RT-PCR detection and fluorescence observation confirmed the expression of the GFP and indicated the existence of the GFP during the chicken embryogenesis and fetal development. The PCR detection and rescue experiments confirmed the episomal state of the pEPI-EGFP in chick embryos and chicks. These results showed that the S/MAR-based vector could function properly in chicken embryos and was a practicable tool combined with the SMGT to study the development of chickens.展开更多
文摘Aim: To confirm the stability of exogenous genes in the generation of transgenic chickens using ejaculated chicken sperm, the deoxyribonuclease (DNase) activity was evaluated in the seminal plasma of ejaculated semen and the stability of DNA was examined by adding lipofection reagents. Methods: A PCR fragment (249 bp) of pEGFPN-1 vector was used as the DNA substrate and was incubated with the seminal plasma at 40 ℃ for 30 min. Then, the whole reaction solution was subjected to agarose gel electrophoresis and the DNA size was evaluated under UV light. Results: The DNA substrate was completely diminished after incubation with seminal plasma. However, the substrate was intact after incubation with heat-treated seminal plasma or incubation with seminal plasma in the presence of 0.5 mmol/L -5 mmol/L EDTA. The substrate was stabilized in the seminal plasma by the addition of commercially available lipofection reagents. Conclusion: The DNase activity is present in the seminal plasma of ejaculated chicken semen. However, DNA is stable in the liposomal-DNA complex.
基金Supported by the National Natural Science Foundation of China(31101033)the Doctoral Research Foundation of Northeast Agricultural University(2009RC56)the Scientific Research Fund of Heilongjiang Provincial Education Department(11551039)
文摘The domesticated chicken has important roles in basic and applied research. The vector based on scaffold matrix attachment region （S/MAR） appears to be sufficient to maintain long-term expression in an episomal state in various mammalian cells. To explore the practical use of episomal vector in transgene technology of agricultural chickens, we fused the S/MAR of the human r-interferon gene into the pEGFP vector and transduced it into chickens by sperm-mediated gene transfer （SMGT）. PCR detection indicated the positive rate of transgene chickens was 60%. The RT-PCR detection and fluorescence observation confirmed the expression of the GFP and indicated the existence of the GFP during the chicken embryogenesis and fetal development. The PCR detection and rescue experiments confirmed the episomal state of the pEPI-EGFP in chick embryos and chicks. These results showed that the S/MAR-based vector could function properly in chicken embryos and was a practicable tool combined with the SMGT to study the development of chickens.
