Highly Efficient Generation of Transgenic Sheep by Lentivirus Accompanying the Alteration of Methylation Status |
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| Authors: | Chenxi Liu Liqin Wang Wenrong Li Xuemei Zhang Yongzhi Tian Ning Zhang Sangang He Tong Chen Juncheng Huang Mingjun Liu |
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| Affiliation: | 1. Xinjiang Laboratory of Animal Biotechnology, Urumqi, Xinjiang, China.; 2. Key Laboratory of Genetics, Breeding and Reproduction of Grass Feeding Livestock, Ministry of Agriculture, Urumqi, Xinjiang, China.; 3. Animal Biotechnology Research Center, Xinjiang Academy of Animal Science, Urumqi, Xinjiang, China.; 4. College of Life Science and Technology, Xinjiang University, Urumqi, Xinjiang, China.; The Walter and Eliza Hall of Medical Research, Australia, |
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| Abstract: | BackgroundLow efficiency of gene transfer and silence of transgene expression are the critical factors hampering the development of transgenic livestock. Recently, transfer of recombinant lentivirus has been demonstrated to be an efficient transgene delivery method in various animals. However, the lentiviral transgenesis and the methylation status of transgene in sheep have not been well addressed.Methodology/Principle FindingsEGFP transgenic sheep were generated by injecting recombinant lentivirus into zygotes. Of the 13 lambs born, 8 carried the EGFP transgene, and its chromosomal integration was identified in all tested tissues. Western blotting showed that GFP was expressed in all transgenic founders and their various tissues. Analysis of CpG methylation status of CMV promoter by bisulfate sequencing unraveled remarkable variation of methylation levels in transgenic sheep. The average methylation levels ranged from 37.6% to 79.1% in the transgenic individuals and 34.7% to 83% in the tested tissues. Correlative analysis of methylation status with GFP expression revealed that the GFP expression level was inversely correlated with methylation density. The similar phenomenon was also observed in tested tissues. Transgene integration determined by Southern blotting presented multiple integrants ranging from 2 to 6 copies in the genome of transgenic sheep.Conclusions/SignificanceInjection of lentiviral transgene into zygotes could be a promising efficient gene delivery system to generate transgenic sheep and achieved widespread transgene expression. The promoter of integrants transferred by lentiviral vector was subjected to dramatic alteration of methylation status and the transgene expression level was inversely correlative with promoter methylation density. Our work illustrated for the first time that generation of transgenic sheep by injecting recombinant lentivirus into zygote could be an efficient tool to improve sheep performance by genetic modification. |
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