The Initial Establishment Of CRISPR/ Cas9 Knockout Method In Mongolian Gerbil

Mongolian gerbils have widely used in many respects as a ―multi-purpose‖ laboratory animals. Studying the pathogenesis, development, treatment and prognosis mechanism of diseases requires a variety of experimental animal models, but the Mongolian gerbil transgenic a nd knockout / knockin animal models have not been established. A convenient and efficient method named CRISPR / Cas9 technology was attempted on the Mongolian gerbil to form Mongolian gerbil transgenic model, which can lay the foundation to study the disea se-related gene functions.In Prokaryotes CRISPR-Cas encoding an adaptive immune system uses to resist viral and plasmids infections. Mmunization is produced by Cas nucleic acid which be mediated by small RNA(crRNAs) digest invading nucleic acid within its genome specific restriction enzyme sites. In Type II CRISPR-Cas system, DNA double-stranded RN A cleavage activity is guided the single enzyme Cas9. By synthetic single guide RNA, Cas9 can reprogram the genome of various organisms to create specific double-stranded DNA breaks, so almost experimental animals can constitute a powerful tool for genetic engineering.Animal cloning and transgenic embryos and other biotechnologies need superovulation provide a sufficient number of eggs, superovulation therefore important for animal embryo research in biotechnology. In order to obtain the best Mongolian gerbil superovulation, we optimized the scheme of Mongolian gerbils superovulation in Mongolian gerbils of Capital Medical University. Based on the analysis of the animal age, the dose and time interval of hormone, we determine an efficient and stable superovulation. Statistics after ovulation found the superovulation optimization scheme: The 6 week old female Mongolian gerbils which injected of 10 IU PMSG and followed by 10 IU hCGin 70 hours later, could get the best effect superovulation. We collected eggs at 16 hour after mate with male gerbils. The ovum pick-up rate reached 80%, the number of oocytes were 32.6±3.0, the number of the fertilized egg developed to 2-cell are 24.8±5.4. We summarizes the optimization scheme of Mongolian gerbil superovulation induced by PMSG and hCG and it supported the foundation for Mongolian gerbils embryo biotechnology.Our target gene sequence by gRNA is designed by search software, found near the shooting site which sequence is the form of 5’-N20NGG-3 ’23bp, and give each site ratings, preliminary screening high activity gRNA. Further testing in vitro gRNA inducing activity detected in vitro enzyme activity better(enzyme activity> 70%) for the screening of the best gRNA.In this project, the selected gRNA sequence inserted into plasmid vector, transfected into DH5α component cells, verified positive bacteria, plasmid extracted, and confirmed the sequence finally. The sequenced plasmid was digested and transcribed into cas9 mRN A and gRNA in vitro. Adjustment cas9 mRNA and gRNA optimal concentration ratio, cas9 mRNA concentration: 100-200ng/μL, gRNA concentration: 10-50 ng / μL, 50 ~ 80% after microinjection of fertilized eggs remain healthy. Training after microinjection healthy 8-cell fertilized egg to a part of the above, genomic DNA, and its knock-detection; another part after microinjectio n healthy fertilized egg, embryo transfer technology through transplanted into pseudopregnant by maternal body within the fallopian tube, until the mice were born, neonatal mouse genomic DNA, knock-off test. Explore a variety of experiments to establish the optimal conditions to lay the foundation for the Mongolian gerbil gene knockout method CRISPR-Cas system.