Construction Of Inner Mongolia Cashmere Goats VEGF Gene Transgenic Cell Lines And Discovery Of A New Forms Of VEGF (VEGF 138)

Objective:To clone the cDNA of VEGF gene in Inner Mongolia Cashmere Goat, Constructing a eukaryotic expression vector pCDsRed2-KV of cashmere goat VEGF164 (vascular endothelial cell growth factor) gene. To transfer pCDsRed2-KV into Inner Mongolia Cashmere goat (Capra hircus) fetal fibroblast (GFb) cells to obtain a transgenic cell clones, which stable expresses red fluorescence and expresses VEGF gene in hair follicle cells specifically. To examin VEGF expression in Inner Mongolia Cashmere goat (Capra hircus) skin cells which tansfected pCDsRed2-KV. In addition, to discovering potential splice variants of transcription products of VEGF gene. To verify the existence of VEGF 138 splice variant in Inner Mongolia Cashmere Goat fetal fibroblast cells. To examine relative abundance of three splice variants of VEGF mRNA in brain, heart, liver, spleen, kidney and lung of Inner Mongolia Cashmere Goats.Method:VEGF gene cDNA was cloned by RT-PCR. The nucleotide sequence was analyzed by bioinformatics. pCDsRed2-KV, a hair-follicle-specific expression vector of VEGF164, was constructed by connecting VEGF164 gene to downstream of KAP6-1 promoter, and then inserting the KAP6-1 promoter-VEGF 164 gene fragment into the basic vector pCDsRed2, which contains a DsRed expression unit. The Inner Mongolia Cashmere goat fetal fibroblast (GFb) cells were transfected with the expression vector by lipofectamineTM2000. Transgenic cell clones were obtained after screening by G418. The recombinant of exogenous DNA was identified by polymerase chain reaction. In order to make sure pCDsRed2-KV is hair-follicle-specific expression vector, we transfected the expression vector into Inner Mongolia Cashmere goat skin cells and detected expression. Expression of VEGF examined by RT-PCR and Western blot assy. Quantitative real-time PCR (Taqman) analysis VEGF 138 mRNA, distribute in Inner Mongolia Cashmere goat (Capra hircus) fetal fibroblast (GFb) cells. The relative abundance of three splice variants of VEGF mRNA, measured in six tissues, by Quantitative real-time PCR.Result:We got CDS fragments of VEGF 164, VEGF 120 and new isoform VEGF138. VEGF138 was discovered for the first time. The VEGF164 gene was 573 bp in length, including an intact ORF which formed by 190 amino acids.26 amino acids of N'end are signal peptidein. The VEGF 138 gene was 495 bp in length, including an intact ORF which formed by 164 amino acids.26 amino acids of N'end are signal peptidein. The VEGF 120 gene was 441 bp in length, including an intact ORF which formed by 146 amino acids.26 amino acids of N' end are signal peptidein. VEGF 164, VEGF 138 and VEGF 138 express in brain, heart, liver, spleen, kidney and lung. VEGF 138 mRNA is less than VEGF 164 and VEGF 120. The sequencing result showed the VEGF 164 gene was connected properly to the downstream of pKAP6-1, then the CMV promoter and the DsRed2 gene in sequence. Exogenous DNA in the cell clones was examined by PCR and the promoter KAP6-1 as well as VEGF164 gene has been integrated into GFb cells genome stably. VEGF mRNA and protein increase significantly in transfected Inner Mongolia Cashmere goat (Capra hircus) skin cells. Conclusion:A hair-follicle-cell-specific expression vector of VEGF 164 gene was constructed successfully and transfered into GFb cells. These data provide a way to obtain the transgenic goat by nuclear transfer in the future. VEGF expression increase apparently in transfected Inner Mongolia Cashmere goat (Capra hircus) skin cells that provide a theory to obtain the transgenic goat. There are three forms of VEGF at least in Inner Mongolia Cashmere goat (Capra hircus). They are VEGF 164, VEGF138 and VEGF120; and VEGF138 was discovered for the first time. Three forms of VEGF express in brain, heart, liver, spleen, kidney and lung. VEGF 138 mRNA is less than VEGF 164 and VEGF 120. This information provides a way to study function of three forms of VEGF.