Transgenic Sheep Of Skeletal Muscle Specific Expression IGF-1

Since the "Dolly" birth, the application of somatic cell nuclear transfer technology in livestock is ascending in the field of animal husbandry. With transgenic somatic cells as donor cells, producing transgenic livestock by nuclear-transfer become the research hotspot.We constructed the muscle-specific insulin-like growth factor I eukaryotic expression vectors, and transfected them into sheep fetal fibroblast cells (SFCs), selected the transgenic cell clones that the exogenous gene stable integration of cells genome. The transgenic cloning embryos of sheep were obtained by somatic cell nuclear transfer technique. The 4-8 cells embryos expressing red fluorescence were transferred to recipients via embryo transfer technology.1 Construction of eukaryotic expression vectors1:Skeletal muscle a-actin gene 5'flank were derived by PCR amplification from the porcine blood DNA, than inserted the SacⅠand SphⅠsite of the p19T-KI vector which contained the ovine IGF-1 cDNA to get a intermediate construction p19T-CI. The vector p19T-CI was digested by SphⅠ+SacⅠto obtain the CI fragment (promoter+IGF-1). The backbone vector pCDsRed2 was linearized by SacⅠ+SalⅠ, then linked with CI fragment. The expression vector was accomplished and named pCICDS;2:The synthetic promoter (SP) was inserted the sacⅠand SphⅠsite of the p19T-KI vector which contained the ovine IGF-1 cDNA to get a intermediate construction. The p19T-SPI was digested by restriction enzyme to obtain the SPI fragment (SP+IGF-1). Then the SPI was inserted into the SacⅠ+SalⅠsite of the backbone vector pCDsRed2 to construct the final expression vector pSPICDS. The evidence from restriction analysis and partial DNA sequencing showed that the two expression vectors were both consistent with anticipation.2 In vitro culture and gene transfection of SFCsSFCs were successfully isolated by attachment of tissue pieces from a fetus of Mongolian sheep. The expression vector pSPICDS and pCICDS were transfected into SFCs genome by Lipofectamine 2000TM. The transgenic SFCs clones were selected by G418 and further selected by red fluorescence. PCR analysed the integration of exogenous gene and the SFCs sexes. Morphology and growth character were observed and recorded, the growth curves of the transgenic SFCs were drawn and the chromosomal analysis of the cells transgenic SFCs within 4th passages were performed. The results showed that the exogenous gene has been integrated into genomes of the two groups of cells respectively, the growth of the transgenic cells was prosperous and the parameters of genetics were normal. The transgenic SFCs should be competent of transgenic cloning.3 The transgenic SFCs nuclear transfer and the embryos transferThe mature oocytes first polar body and nucleus were aspirated through micromanipulation method, and then transferred the transgenic donor cells to the Enucleated oocyte. The donor cell-enucleated oocyte complex was fused by electronic methods.5μM IA23187 for 5min plus 2mM 6-DMAP for 4hr to activate the fusion oocyte. Transgenic embryos were PCR analysed the integration of exogenous gene and observed the expression of red fluorescent protein by laser confocal.4 to 8 cells of transgenic embryos which were transplanted into the oviduct of recipient sheep. Result showed that maturation rate of the oocyte was 67%, the fusion rate was 61.7%, the cleavage rates was 79.5%,8cells rate was 57%, blastocyst rate was 13%; Exogenous gene have been integrated into the transgenic embryos genomes and embryos expressed the red fluorescent protein. Transgenic embryos had been transplanted into 27 recipient sheep. Four recipient sheep were pregnancy, one of them came to birth one female lamb, and the rest were abortion.