动物造模,CRISPR/Cas9 z-bio 2022-03-09 596

　　Objective: To construct a taurine transporter knockout rat by CRISPR/Cas9 technology, breed and identify it, and provide a stable rat model for studying the effect of taurine deficiency on nervous system diseases.

　　Methods: Aiming at exon 5 of Slc6a6 gene, a guide RNA (single-guide RNA, sgRNA) was designed to mediate the specific binding of Cas9 nuclease to target DNA, and cleave genomic DNA, and the cleaved DNA was recombined and repaired. To achieve gene knockout. The genotype of neonatal rats was detected by genotype identification and sequencing analysis. Real-time PCR, Western blot technology and immunohistochemistry were used to analyze the taurine transporter in rat brain tissue. , TauT) mRNA and protein expression to establish a Slc6a6 knockout rat model.

　　Results: There were 21 Slc6a6 knockout homozygotes (TauT-/-), 54 heterozygotes (TauT+/-), and 27 negative (TauT+/+) in the F3 generation. The homozygous rate of the F3 generation was about 20.59%. In line with Mendelian inheritance law. Slc6a6 knockout homozygous rats basically did not express the mRNA level in brain tissue, and the expression level of TauT protein was significantly lower than that of littermate-negative rats.

　　Conclusion: In this study, the CRISPR/Cas9 system was used to knock out the Slc6a6 gene, and the Slc6a6 knockout rat model was successfully constructed.