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
Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Such approach is referred to as e...Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Such approach is referred to as either male germ cell mediated gene transfer （MGCMGT） or female germ cell mediated gene transfer （FGCMGT） technique. Sperm-mediated gene transfer （SMGT）, including its alternative method, testis-mediated gene transfer （TMGT）, becomes an established and reliable method for transgenesis. They have been extensively used for producing transgenic animals. The newly developed approach of FGCMGT, ovary-mediated gene transfer （OMGT） is also a novel and useful tool for efficient transgenesis. This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques, methods developed and mechanisms of nucleic acid uptake by germ cells.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 Peking University People’s Hospital Research and Development Foundation(No.RDB2007-03)
文摘Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Such approach is referred to as either male germ cell mediated gene transfer （MGCMGT） or female germ cell mediated gene transfer （FGCMGT） technique. Sperm-mediated gene transfer （SMGT）, including its alternative method, testis-mediated gene transfer （TMGT）, becomes an established and reliable method for transgenesis. They have been extensively used for producing transgenic animals. The newly developed approach of FGCMGT, ovary-mediated gene transfer （OMGT） is also a novel and useful tool for efficient transgenesis. This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques, methods developed and mechanisms of nucleic acid uptake by germ cells.
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.
