Establishment of a Technique to PCR Detection about Follistatin Transgenic Sheep by Injection of Lentivirus

[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.

Production of Brown/Yellow Patches in the SLC7A11 Transgenic Sheep via Testicular Injection of Transgene

The gene, SLC7All, which encodes the solute carrier family 7 member 11 （anionic amino acid transporter light chain, xCT）, has been reported to be implicated in multiple processes such as in pheomelanin production, cell proliferation and migration, Kaposi＇s sarcoma herpesvirus （KSHV） entry into the host cells, learning and memory. Its involvement in cancer cell proliferation and metastasis has been widely studied. Its role in pheomelanogenesis is likely conserved in sheep. The full-length cDNA of sheep SLC7A11 was cloned from sheep skin fibroblasts for evaluating its role in regulating sheep coat color. The complete open reading frame of sheep xCT （sxCT） is 1512 bp in length, encoding a 503 amino acid polypeptide. We explored its function on pheomelanogenesis in vitro and in vivo. In the melan-a non-agouti mouse melanocytes that mainly produce eumelanin, overexpressed sxCT reduced the content of eumelanin. Using a testicular injection transgenic method, sxCT-transgenic sheep were generated and exhibited patches of brown/yellow coat, suggesting that sxCT can be selectively expressed to increase the pheomelanin production in wool. Our studies suggest that testicular injection of transgene can be used to genetically modify sheep coat color.