The Construction Of Expression Vector Of Fat-1and Establishment Of Transgenic Suffolk Sheep Fibroblast Cell Line

Objective:ω-3PUFAs palys an important role in maintain normal development, growth and disease prevention of living organisms. Invivo ω-6/ω-3PUFAs level cannot be balanced to1:1as human being is lack of the dehydrogenase gene in transforming ω-6PUFAs to ω-3PUFAs. Therefore, to ensure that the normal organism function and its stability, a certain amount of ω-3PUFAs must be consumed every day. At present, deep-sea fishes are the main resources of ω-3PUFAs in daily food. As ω-3PUFAs source is very narrow, human body’s ω-6PUFAs level are too high and ω-3PUFAslevel are severity low, which results ina postitive function in enhancing infants intelligence developing and preventing cardiovascular and cerebrovascular diseases and cancercan not be executed. Thus, we devote to creating one new breed sheep, which can expressing dehydrogenase gene Fat-1of ω-3PUFAs, to obtain the high quality mutton with high level of ω-3PUFAs.Methods:1The construction of pUbB-oFat-polyA-CMV-DsRed and establishment of transgenic sheep fibroblast cell lineThe male ovine fibroblast cells were isolated and cultured by attachment of tissue block from18-month old ovine. UbB was obtained by PCR, Fat-1gene from Caenorhabdit elegans was optimized base on ovis aries codon, and GBHpolyA was from pcDNA3.1.These donor sequences were orderly inserted into MCS of pCMV-DsRed2-1vector. The recombinant vector (pUbB-oFat-polyA-CMV-DsRed) was analysed by endonucleases digestion and sequencing. The linearized vector was transfected into sheep fibroblast with liposome. The positive cell was selected with G418and red fluorescence signal,and furtherly confirmed by PCR and RT-PCR,components of PUFAs were detected by HPLC.2.The preparation of transgenic sheepReconstructed embryos, Fat-1transfection ovine fibroblasts and matured ovine oocytes with nucleus kicked were used as donor cells and recipient cells, respectively. After fusion, activation and development,2cell stage of reconstructed embryos were obtainedand transferred into the uterus of31synchronous processing receptor sheep.6-7reconstructed embryos per sheep. Detection the newborns by the PCR, analysis of cell chromosome number of cloen sheep. Display all have normal diploid karyotypeResults:The results showed that corrcet recombinant vector was obtained,and Fat-1gene could been properly expressed in sheep fibroblast. The ω-3PUFAs were greatly increased from11.48%to24.41%and simultaneously the ω-6PUFAs decreased from88.52%to75.59%.A significantly reduction of ω-6/ω-3PUFAs ratio was observed from7.82±0.18to3.10±0.03(P<0.01). At last, after nuclear transfer and embryonic transplantation, there are3of31sheep pregnancy, the pregnancy rate was about10%, and gave birth to3sheep totally. Primary detection by the PCR confirmed2of3newborn were transgenic sheep. Detection the newborns by the PCR, analysis of cell chromosome number of cloen sheep. Display3sheep have normal diploid karyotype. Conclusion:These results demonstrate that the expression vector was constructed successfully, and the sheep fibroblast line was obtained, which could encode unsaturated fatty acid desaturase. At last, we had received2transgenic clone sheep. Furthermore,this study may pave the way to generate transgenic sheep by somatic cell nuclear transfer technology.