Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover,...Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.展开更多
Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( ...Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.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
基金supported by the National Major Special Project on New Varieties Cultivation for Transgenic Organisms,China(2014ZX08006-005 and 2014ZX0800950B)
文摘Homeobox A10(Hoxa10) gene is one of the most important candidate genes associated with the reproductive performance of humans and mice. Overexpression of Hoxa10 in mouse endometrium can increase litter size. Moreover, Hoxa10 plays a key role in regulating the embryo implantation of sows. This study aimed to generate transgenic pigs using Hoxa10 via somatic cell nuclear transfer(SCNT). We established seven Hoxa10-transgenic cell lines, and two of the cell lines were selected as nuclear donors for the transfer. A total of 1 270 cloned embryos were generated and transferred to five surrogate mothers(Landrace×Yorkshire). Eight cloned male piglets were produced including one with cryptorchidism. Six transgenic piglets grew up healthy and produced 56 offspring. Finally, we obtained six transgenic male pigs and 26 transgenic positive offspring that can be used to further study the regulatory mechanism of Hoxa10 on the reproductive performance of pigs.
基金Scientific Research Project for High Schools of the Educational Department of Liaoning Province,China(No.2008643)
文摘Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.
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.
