The Study On The Cloned Sheep Derived From Fat-1 Gene Transferred Somatic Cell

Fat-1 gene encodingω-3 polyunsaturated fatty acids (PUFAs) desaturase, can convert polyunsaturated fatty acids fromω-6 toω-3 form. The aim of study present here was to obtain transgenic sheeps, which contained abundantω-3 PUFAs in their muscles, through transferring the fat-1 gene into ovine genome and then production the transgenic cloned sheep by animal cloning technology. By means of our predominant sheep species, we optimized the construction and embryo transferring system of ovine cloned embryo, established male ovine fetal fibroblast cell line, cloned the fat-1 gene from Caenorhabditis elegans and constructed the expression vector containing purpose gene. In vitro cultured ovine fetal fibroblast cells were transfected with this vector. The stable transfected cell lines were established through G418 screening, and the transgenic cells were identified by PCR. Using the nuclei from selected donor cells, ovine embryos were produced via nuclear transfer technology and the reconstructed embryos were transferred to recipients via embryo transfer technology. Transgenic generations to be identified and detected the relative amount and ratio ofω-6 andω-3 PUFAs in the muscle after the birth, so that to obtain transgenic sheep with new varieties of health functions.1. Optimization of sheep somatic cell cloning systemThe aim of this experiment is to optimize transferring system of somatic cell cloned embryos on developmental stages of reconstruction embryo. Three female ovine ear skin fibroblast cell lines were isolated and cultured by attachment of tissue pieces and the exploration divided into three stages.The first stage, we constructed 272 cloned embryo from No. 1 cell line, than transferred into 17 recipient female sheeps when the embryos developed in the morula stage in vitro. There is one pregnancy and the pregnancy rate was 5.88%. One lamb was born, and it was identified as Cloned sheep.The second phase we constructed 683 cloned embryo from No. 2 cell line, than transferred into 35 recipients when the embryos developed in different stages in vitro. The results showed that, there was a pregnancy in 3 recipients which were transferred the 1-cell embryos, and born one lamb which was identified as Cloned sheep.The third phase we constructed 197 cloned embryo from No. 3 cell line, than transferred into 11 recipients when the embryos developed in 1-cell stage in vitro. The results showed that, there were two pregnancy and the pregnancy rate was 18.18%. Three lambs were born and the lambing rate was 27.27%. This result effectively repeated the second phase of experiments. The cloned sheep was wildly paired with ram after it developed to sexual and physical maturity. The adult somatic cell cloned sheep delivered two twins. The results showed that we obtained cloned sheep with normal reproductive and feeding ability for offsprings , and further prove that the laboratory had established a relatively stable sheep somatic cell cloning technology system.2. Establishment of transgenic somatic cell cloning systemThe male ovine fetal fibroblast cells were isolated and cultured by attachment of tissue pieces from 40d old ovine fetal. We cloned the fat-1 gene from Caenorhabditis elegans, optimized the fat-1 gene sequence according to ovine hobby codons, and synthesis the optimized fat-1 gene sequence. Then we constructed pTFK-c and pTFK-o eukaryotic expression vectors. Sheep fetal fibroblasts were transfected by pTFK-o, screened by G418, and identified by PCR. The results showed that 5 cell lines genome had integrated the target gene in 7 clones obtained. The chromosome number analysis of the transgenic cell showed that the normal karyotype was 69.77% (2n = 54), so the transgenic ovine fetal fibroblast cells gave a good fit to transgenic cloning manipulation. The transgenic ovine fetal fibroblast cell line 7 as nuclear donor cell and enucleated oocytes served as recipient cytoplasm to produce nuclear transferred embryos. Totally 4881 oocytes were collected by aspiration, 72.87% of them were matured after 18 hours culture and 3557 oocytes were enucleated and 3305 enucleated oocytes were treated for electrofusion with donor cells. Among them, 3040(58.4%) couplets were fused and the fusion rate was 91.39%. Totally 2909 reconstructed 1-cell embryos divided into three groups were transferred into 230 recipients. Group 1 transferred into 146 recipients, and there were 5 pregnant sheeps and pregnancy rate was 3.42%, lambing 5 and lambing rate was 3.42%. Group 2 transferred into 60 recipients, 18 pregnancy and the pregnancy rate was 30%, 19 lambing and lambing rate was 31.67%. Group 3 transferred into 24 sheeps, 5 pregnancy and pregnancy rate was 20.83%, 6 lambing and lambing rate of was 25%. The results of last 2 groups further optimized the somatic cell cloning technology system.3. Birth and identification of fat-1 transferred cloned sheepsWe obtained 30 lambs and 19 survived. There were 27 ovine genome contains fat-1 gene in 29 lambs. The detection results of relative amount ofω-6 andω-3PUFAs in cloned sheep muscle tissue showed that, there were 18 in the 23 sheeps expressed fatty acid desaturase, theω-6/ω-3 PUFAs ratios of them were lower, and the average was 16.45. The average of relative amount aboutω-3 PUFAs was 0.194, which increased 31.97% compared to that of control rams (0.147). This work effective improved theω-6 andω-3 PUFAs relative proportion and content.