The Research On CRISPR/Cas9 Mediated VEGF Site-specific Integration In Cashmere Goat

The Inner Mongolia Alba white cashmere goat is a unique biological resource in our country,which is one of the species with the highest production and the best fiber quality of cashmere in the world currently through long-term natural selection and artificialbreeding.It is cashmere goat breeding target to improve cashmere production in quantity and quality.VEGF gene modified cashmere goats have been obtained in our laborator and the cashmere production has been improved significantly,which shows a good prospect of breeding.In recent years,the specific traits genetic constitution can be changed quickly through genome editing techniques to obtain the required species.In this research gRNA expression,and VEGF gene knock-in vectors at site of CCR5 were constructed by CRISPR/cas9 genome editing technique.CFFCs were transfected with those vectors by electroporation,the positive cells of VEGF knock-in at CCR5 were selected and used as donor cells for nuclear transfer to produce VEGF knock-in gaots.These results could provide sufficient evidences of the merit of using CRISPR/cas9 approach for the generation of gene-modified goats displaying high cashmere production.1.Vector ConstructionFour gRNAs vectors at CCR5 of exon 2 were constructed successfully,the mutation efficiency of CCR5-gRNA4 was detected through survery enzyme mutation detection and the other 3 had no mutation,so gRNA expression vector was CCR5-gRNA4.VEGF knock-in vector in CCR5 gene locus(P1-KV-polyA-P2)was constructed successfully as well.2.Selection of positive monoclonal cellsP1-KV-polyA-P2,CCR5-gRNA4,and hCas9 were transfected into CFFCs via electroporation by the most appropriate transfection efficiency and proportion.After 48 h electroporation,positive monoclonal cells were screened by flow cytometry or selected by glass pipette to established monoclonal cell lines.Positive monoclonal cell lines were selected by PCR amplifying and sequencing successfully.3.Preparation of VEGF site-specific integration cashmere goatsIn this research,cashmere goat was generated by somatic cell nuclear transfer and pronuclear microinjection respectively.747 positive monoclonal cell reconstituted embryos were produced through somatic cell nuclear transfer and transferred into 150 recipient female goats.and 3 kids were born and 1 died after 7 days.30 embryos microinjected constructed vectors were transferred into 15 recipient female goats.and 8 kids were born.Two kids obtained by SCNT were detected VEGF site-specific integration by PCR.While none of the kids got by microinjection was VEGF site-specific integration goat.The mRNA expression of two VEGF site-specific integration goats was detected by real-time PCR.It showed that the expression of VEGF mRA in their skin tissues was significant difference with NT,respectively 1.68 times and 1.94 times than that of NC.Protein expression of VEGF was detected by western blot.The results showed that the protein expression level of VEGF site-specific integration goat increased significantly.In conclusion,VEGF site-specific integration cashmere goats had been produced successfully in this research,and could be used as breeding material in future.