Establishment A Preliminary Study Of Gene Editing Technique In Rabbit Using CRISPR/Cas9 System

The use of gene knockout experimental animals we can directly detect the pathogenesis of disease development process,revealing the gene,cell structure and the relationship between the symptoms.Transgenic rabbits have become an important tool for gene function research,the establishment of human disease models and the production of high-value medicinal proteins.The mouse Rosa26 locus has been widely used for site-directed integration of foreign genes.In recent years,the Rosa26 locus of pigs has also been studied by relevant scholars and successfully cultivated transgenic pigs.There was a safe locus on chromosome 11 in mice.In 2010,the site was successfully isolated and identified by Stanford's research team,named hipp11,the H11 site.This locus is located between exon 9 of the Drg1 gene and exon 19 of the Eif4enif1 gene,with a size of about 5 kb.Because it is located in the interval sequence,so there is a high security,not easily silenced by genes,can be expressed in a variety of cells.Object:In this study,the rabbit CRISPR/Cas9 system was selected as the object of study.The aim was to improve the rabbit genetic manipulation system and establish a mature and efficient rabbit gene knockout platform,which was of great significance to the promotion and application of genetic engineering rabbits.It provides new methods and new methods for gene function research,human disease animal model and high value protein mammary gland bioreactor development.Methods:(1)Using the CRISPR/Cas9 gene editing system,the knockout vector was constructed according to the MSTN gene of rabbits,and the knockout efficiency was verified at the cell level.(2)The Rosa26 and H11 loci were identified by bioinformatics to construct knockout vectors and homologous targeting vectors of Rosa26 and H11 loci.The knockout efficiency was verified at the cellular level and the integrated cell line was constructed.(3)Comparison of different doses of PMSG super-row effect,select the best dose of PMSG and FSH super-row were compared to select their own laboratory super-row program.Results:(1)Design targeting MSTN genome gRNA fragment 2,to build the corresponding knockout vector 2.A CRISPR vector targeting MSTN gene was screened and the knockout efficiency was 20%.(2)Rabbit H11 loci were identified on the chromosome 21 of rabbits,and two corresponding knockout vectors targeting H11 and Rosa6 were constructed.The targeting vectors of H11 and Rosa26 were constructed respectively.By fluorescence observation,drug screening,Combined with PCR technology,successfully obtained H11 and Rosa26 site integration Puro gene and ZsGreen1 gene cell line.(3)In the experimental group,the number of fertilized eggs was the highest in the PMSG rabbits injected with 80 IU PMSG.The difference between the appropriate PMSG dose(80 IU / mouse)and the superovulation effect of FSH on rabbits was not significant.FSH + HCG was selected the best ultra-row regimen for this experiment.Conclusion:(1)In this study,an efficient gene knockout and site-directed integration system was established at the cellular level according to CRISPR / Cas9 gene editing technology,which laid the foundation for the safe and efficient preparation of transgenic rabbits in the future.(2)The ratio of PMSG dose(80 IU / mouse)to FSH was better than that of the control group(P <0.05).The difference was not significant.Select FSH + HCG as the best ultra-row regimen for this experiment.To establish a mature super-row system for the subsequent preparation of transgenic rabbits lay the foundation.