The Production Of Transgenic Sheep Embryos Transfected With Green Fluorescence Protein And Augmenter Of Liver Regeneration Genes

In this research augmenter of liver regeneration (ALR)gene was transferred in sheep embryos, and an efficient screening technique has been developed to detect transgenic sheep embryos using enhanced green fluorescence protein (EGFP) as genetic marker. The eukaryotic expression vectors were used for the sheep fetal fibroblast cells (sFFCs) transfection. An effective method to select transgenic embryos formed by nuclear transfer using tranfected donor cells was established. Introducing of this technique into the production of human augmenter of liver regeneration using transgenic sheep will not only reduce the amount of recipient sheep needed and the cost but also increase the efficiency of gene transfection1. Construction of containing both EGFP marker and ALR geneHuman ALR and sheep BLG genes were amplified from human and sheep genomic DNA using PCR respectively. The PCR technique was also used to produce CMV, EGFP and SV40PolyA sequence from plasmid pEGFP-Cl.Three vectors were constructed:(1) The breast specific expression of ALR was controlled by BLG promoter, and the non-specific expression of EGFP gene was controlled by CMV dual promoter system (pGME-BAE). (2) ALR gene was also inserted to bicistronic plasmid p!RES2-EGFP to produce ALR, EGFP gene bicistronic eukaryotic expression vector (pIRES-EGFP/ALR). (3) ALR gene was inserted into pEGFP-Cl plasmid to construct ALR and EGFP co-expression vector pEGFP/ALR. Both PCR and enzyme digestion analysis confirmed all the three constructed plasmids were correct.2. Sheep Fetal Fibroblast cell culture and transfectionsFFCs had been successfully isolated from six 30-day-old sheep fetals and cultured in DMEM/F12 media. The sensitivity studies of sheep fetal fibroblastsagainst G418 concentration in DMEM/F12 media indicated as high as 800ug/ml of G418; and the concentration of G418 can be reduced to 300ug/ml during the cell maintenance stage. Sheep fetal fibrobasts were transfected with pEGFP-Cl by using both LipofectAMINE?and Fegene-6 substance. The impacts of DNA purity, DNA concentration, concentration of transfection agents, exposure time as well as incubation time on transfection efficiency had been carefully compared. The best results were obtained when sheep fetal fibroblasts were incubated with lOul LipofectAMINE?and 2ug DNA for 8 hrs. The transfection rate was 42 cells in a visual field, and fugene transfection rate was 19 cells in a visual field. LipofectAMINE?transfection method developed in this paper can be easily adapted to most research labs and provides a basic reference for transfection of sheep fetal fibroblasts with target gene.3. Biological impact of EGFP and sex gene identification on sheep fetal fibroblastsSheep fetal fibroblasts were transfected with pEGFP-Cl usingLipofectAMINE . The transfected cells were incubated in DMEM/F12 media containing G418 for 12 days. Cells which GFP expression were picked up and continued culture for another 23 days. There was no obverse difference had been found between transfected cell and the same age normal sheep fetal fibroblasts in term of cell anatomy, growth curve as well as chromosome analysis.The results of sex gene identification of six transfected sheep fetal fibroblasts were also match with the results of chromosome analysis. Therefore, the sexing GFP-transfected sheep fetal fibroblasts can be used as nuclear donor in the process of produce transgenic sheep using nuclear transfer.4. Transfection and expression of sheep fetal fibroblasts with pEGFP/ALRSheep fetal fibroblasts were transfected with pEGFP-Cl/ALR using LipofectAMINE? The transfected cells were incubated in the selected media containing 800ug/ml G418, Totally 38 clones were formed 10 days later, 23 out of 38 clones were green fluorescence positive, and 15 out of 38 clons were no-fluorescence negative. Among them, 3 positive clones and 3 negative clons demonstrated with great growth potential were selected for further studies. Both ALR and NEO genes were detected in these fluorescence positive clones...