Production Of Fat-1Transgenic Cloned Sheep And Methylation Analysis Of Their Promoter Regions

This study was designed to produce fat-1transgenic sheep by using two different expression vector driven by CMV or CAG promoters, respectively. The n-3unsaturated fatty acid desaturase gene (fat-1) was isolated roundworm and built as expression vectors under the control of CMV or CAG promoter. The vectors were then transfected into sheep fetal fibroblast cells respectively and the positive cells were used as donors to create cloned animals by nuclear transfer. Analyses of the transgenic sheep showed that two different expression patterns were observed. High methylations of CMV promoters in a various transgenic tissues probably resulted in the fat-1gene silence in the sheep. However, CAG methylations in tissues almost did not affect the transgenic fat-1expression.1. Preparation of transgenic fat-1gene cloned sheep driven by CMV promoterThe fat-1gene was isolated from total RNAs of Caenorhabditis elegans and ligated into MCS of plasmid pIRES2-EGFP driven by CMV promoter, making it co-expressing with green fluorescent protein. Sheep fetal fibroblast cells were transfected and screened. The cells which expressed exogenous genes stably was used as donor cells for SCNT. Then transgenic reconstructed embryos were transferred into oviducts of synchronization Mongolian sheep. Five months later, two cloned lambs developed to the term, named H001and H002. Although PCR and Southern blots analyses using tissues from the two lambs derived from CMV vectors indicated integration of fat-1gene into the genome, fat-1mRNAs were not detected by RT-PCR. Furthermore, muscle tissues from H001, H002and the control were pulverized and subjected to lipid composition analysis using mass spectrometer. There was no significant difference in the fatty acid composition profiles between the transgenic clones and the non-transgenic control.2. Methylation analysis of transgenic pCMV-fatl cloned sheepTransfected cells, resulted embryos and the tissues of H001and H002were collected to analyze methylation status of CMV promoter region. After prediction of online software, CMV promoter had three CpG islands and the longest one was chosen as target of bisulfite sequencing. A338bp DNA fragment in a CpG island with19CpG sites was amplified with two pairs of BSP nested PCR primers. All the purified DNA fragments were ligated into cloning vector pMD-19T and were sequenced. The results demonstrated that CMV cells and resulted embryos were almost unmethylated, whereas all the tissue samples, including the heart, lungs, liver, muscle, kidney, brain, skin, testis of H001and H002, were highly methylated, with the methylation ratio of92.6%on average. Moreover, methylation and expression of the SV40promoter in pCMV-fatl transgene were also analyzed. The results showed that there was no methylation in CMV cells and resulted embryos, but all the analyzed tissues were in high methylation status and the methylation ratio on average was79.4%. The Expression of Neor gene was inversely related to its methylation level. The conclusion was the same as that in fat-1expression driven by CMV promoter.3. Preparation and methylation of transgenic fat-1gene cloned sheep driven by CAG promoterpCAG-fatlvector was constructed based on pCMV-fat1. CMV promoter was removed from pCMV-fatl and CAG promoter from pCAGEN were ligated into remaining fragment. Compared with original vector, other expression elements were same except promoter region. After SCNT using CAG positive cell line as donor cells,4301-to2-cell embryos were transferred into oviducts of the90sheep receptors. Fifty days later, the pregnancy rate was8.9%with ultrasonography detection. About70days after gestation, two fetuses were isolated from surrogate mother with caesarean section, named N001and N002. Fetal fibroblast cell lines were prepared with the back tissues of the two fetuses. PCR and RT-PCR showed fat-1gene positive. The rates of cells with green fluorescence with flow cytometry detection were14.90%and8.68%, respectively. The tissues of two fetal sheep, including heart, lung, liver and kidney, showed fat-1gene positive by RT-PCR analysis. Then, two CAG cloned lambs developed to the term, named N003and N004. Adult fibroblast cell lines were prepared with the ear tissues of the two lambs and N003cell line emitted green fluorescence. PCR and RT-PCR of N003cells showed fat-1gene positive.Transfected cells, resulted embryos and the tissues of N001and N002were collected to analyze methylation status of CAG promoter region. A276bp DNA fragment located in a CpG island with32CpG sites was amplified with two pairs of BSP nested PCR primers. All the purified DNA fragments were ligated into cloning vector pMD-19T and were sequenced. The results demonstrated that CAG cells and resulted embryos were almost unmethylated, whereas all the tissue samples, including fetal fibroblast cell, the heart, lungs, liver, kidney of N001and N002, were highly methylated with the methylation ratio of95.3%on average except N001liver with a moderate rate,66.2%. These data implied that although CAG promoter was highly methylated in transgenic animals, gene expression driven by it was not affected.