Establishment And Identification Of Conditional Knockout Rat Model Of Drp1 Gene

Objective:Based on Cre/Lox P system,CRISPR/Cas9 technology was used to construct a flox heterozygous rat model that could carry out conditional knockout(CKO)of liver-specific dynamin-related protein 1(Drpl)gene.This study provides an experimental basis for further study of the biological function of Drp1.Methods:1.By using NCBI Genebank to find the rat gene encoding Dnm1 l,also known as Drp1,the gene sequence of Drp1 was analyzed,gene knockout targets were designed,and g RNA target sequences were determined;Simultaneously construct homologous recombination vectors and prepare sg RNA and Cas9 proteins.2.The mixture of prepared sg RNA,Cas9 protein and homologous recombinant vector plasmid was directly injected into the single cell zygote by microinjection method.The injected zygote was transferred to the oviduct of surrogate SD female rats,waiting for the birth of the offspring F0.The F0 generation of positive rats was identified by PCR and DNA sequencing.3.The F0 generation of positive rats was born for 8 weeks,and they were cooped with wild-type SD rats for 8 weeks.After 1 week of birth,the offspring were identified for genotype using tail PCR,gene sequencing,and Southern Blot assay techniques.The offspring were screened to obtain the F1 generation of positive rats.Results:Successfully constructed a targeting vector,which was identified by PCR to be consistent with the predetermined base number;The target gene rat F0 was successfully obtained using CRISPR/Cas9 technology,and stable inheritance was achieved after mating with wild-type SD rats.Ten positive F1 generation flox heterozygous rats were obtained,which were identified correctly by PCR and sequencing.There was no significant abnormality in the phenotype of Drp1 gene flox heterozygous rats.Conclusions:We have successfully constructed a flox heterozygous rat model with conditional knockout of the Drp1 gene for the first time,laying the foundation for the future preparation of tissue specific knockout rat models of the Drp1 gene,and providing a reliable in vivo experimental model for in-depth research on the function of the Drp1 gene and the discovery of molecular mechanisms underlying related diseases.