Preparetion Of Cashmere Goats Integrated VEGF Gene At FGF5 Site Via CRISPR/Cas9

Cashmere is a kind of precious special animal fiber in high economic value.Arbas Cashmere Goat is an excellent local breed formed by long-term natural selection in cashmere and meat dual-purpose,and is renowned for superior quality cashmere.But it is necessary to breed new varieties of cashmere goat to improve cashmere production.Compared with traditional breeding,modern biotechnology could greatly shorten the breeding,and provides new technical ways for the cultivation of new varieties of agricultural organisms.VEGF acts on vascular endothelial cells,and promotes the proliferation;and metastasis of vascular endothelial cells,and promotes vascular permeability and angiogenesis.It has the function of inducing hair follicle morphogenesis and regulating hair growth cycle.The over-expression of this factor in the skin of cashmere goats can increase the yield of cashmere.FGF5(Fibroblast growth factor 5)has a negative regulatory effect on hair growth.FGF5 knocked out in cashmere goat can prolong the growth period of hair and delay to entry degenerative stage,thus the growth time of hair is prolonged and a new character of long hair is obtained.In this research VEGF gene was integrated at FGF5 site of cashmere goat fetal fibroblast by CRISPR/Cas9,achieved an objective of VEGF gene at FGF5 site of cashmere goat fetal fibroblast,realizing VEGF gene over expression and FGF5 gene knock out at the same time.Then the cashmere goats integrated VEGF gene at FGF5 site were obtained by somatic cell nuclear transfer,so that to provide breeding materials for breeding of new cashmere goat varieties.1.Construction of VEGF gene targeted integration vectorsCas9/gRNA co-expression vector was constructed successfully,and its mutation efficiency was evaluated by surveyor mutation detection kit.And then,the VEGF targeting vector which was consisted of FGF5 first exon upstream fragment,Kap6.1 promoter,VEGF gene,polyA,and FGF5 first exon downstream fragment was constructed successfully.2.The acquisition of monoclonal cells integrated VEGF gene at FGF5 siteThe VEGF targeting carrier of enzyme tangent and the Cas9/gRNA co-expression vector were transferred into the cashmere goat fetal fibroblasts by electrorotation.After 48 h single cells were inoculated into 96 well plates by flow cytometry.The genome of obtained monoclonal cells was extracted for PCR amplification and sequencing to identify whether VEGF gene has been knocked-in or not.In this experiment,total 121 monoclonal cells were obtained,5 strains of them were VEGF gene integrated after identification,and the efficiency of integration was 4.1%.3.The production of cashmere goats integrated VEGF gene at FGF5 siteTotally 1824 oocytes were collected from 463 ovaries by mechanical cutting method,and 936 oocytes were matured in vitro,and the maturation rate was 51.3%.The nuclei and polar bodies of matured oocytes were removed and the fetal fibroblasts integrated VEGF gene at FGF5 site were injected by somatic cell nuclear transfer,and 848 reconstructed embryos were obtained.After two times of electric fusion,591 reconstructed embryos successfully fused and the fusion rate was 69.7%.After fusion,the reconstructive embryos were cultured in vitro,of which 367 were cleavage,and the cleavage rate was 62.1%.The reconstructed embryos after cleavage were transplanted into oviduct of 131 recipient goats,2 kids was obtained the next year.Blood samples from kids were used to extract the genome for preliminary identification,and one of them was identified as integrated VEGF gene in FGF5 site.In this experiment,cashmere goats integrated VEGF gene at FGF5 site were obtained by CRISPR/Cas9 technology,which provided a possibility for the cultivation of high quality and quality cashmere goats.