[Objective] The aim was to establish a stable detection method of lentivirus transgenic sheep at DNA level.[Method] The cotyledons,umbilical cord,tail tissue of newborn transgenic lambs and the body tissues of dead la...[Objective] The aim was to establish a stable detection method of lentivirus transgenic sheep at DNA level.[Method] The cotyledons,umbilical cord,tail tissue of newborn transgenic lambs and the body tissues of dead lambs were collected and used to extract DNA for PCR with primers designed for N＋D1 fragment of follistatin gene.At the same time,we detected the CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector.The body tissues of dead lambs and muscle tissues of transgenic lambs in vivo were used to extract RNA for RT-PCR.[Result]The results showed that the DNA Extraction Kit was faster and more efficient than conventional method in extracting DNA and the DNA extracted with kit was easier to be amplified than that with conventional method.In order to avoid false positive caused by the interference of endogenous gene,the primers were designed for amplifying the combination of upstream of vector gene and downstream of target gene,increasing the specificity of detection.Tail tissue of newborn transgenic lambs could be used for detection and the detected results were reliable and accurate.The detection of CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector provided a data support for the biological safety of transgenic animals and verify the detected results of target gene of transgenic lambs.The transcription products of RNA extracted from three of the lambs were not detected.[Conclusion] The PCR method established in our research for detecting transgenic sheep was efficient,fast and accurate.It would provide experimental basis for further detection at protein level,lay a foundation for the establishment of multi-level and systematic detection method of transgenic sheep and provide a stable technology platform for safety monitoring of transgenic sheep.展开更多
基金Supported by National Major Transgenic Project (2013ZX08008-003-04,2013ZX08010004-009)~~
文摘[Objective] The aim was to establish a stable detection method of lentivirus transgenic sheep at DNA level.[Method] The cotyledons,umbilical cord,tail tissue of newborn transgenic lambs and the body tissues of dead lambs were collected and used to extract DNA for PCR with primers designed for N＋D1 fragment of follistatin gene.At the same time,we detected the CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector.The body tissues of dead lambs and muscle tissues of transgenic lambs in vivo were used to extract RNA for RT-PCR.[Result]The results showed that the DNA Extraction Kit was faster and more efficient than conventional method in extracting DNA and the DNA extracted with kit was easier to be amplified than that with conventional method.In order to avoid false positive caused by the interference of endogenous gene,the primers were designed for amplifying the combination of upstream of vector gene and downstream of target gene,increasing the specificity of detection.Tail tissue of newborn transgenic lambs could be used for detection and the detected results were reliable and accurate.The detection of CMV promoter,5＇-LTR and so many other structure elements of lentiviral vector provided a data support for the biological safety of transgenic animals and verify the detected results of target gene of transgenic lambs.The transcription products of RNA extracted from three of the lambs were not detected.[Conclusion] The PCR method established in our research for detecting transgenic sheep was efficient,fast and accurate.It would provide experimental basis for further detection at protein level,lay a foundation for the establishment of multi-level and systematic detection method of transgenic sheep and provide a stable technology platform for safety monitoring of transgenic sheep.
