动物造模,CRISPR/Cas9 z-bio 2021-11-12 301

　　Purpose: Use CRISPR/Cas9 technology to construct taurine transporter gene knockout rats, reproduce and identify them, and provide a stable rat model for studying the effects of taurine deficiency on neurological diseases.

　　Method: Aiming at the 5th exon of the Slc6a6 gene, design a guide RNA (single-guide RNA, sgRNA) to mediate the specific binding of Cas9 nuclease to the target DNA, and cut the genomic DNA, and the cut DNA is recombined and repaired, thereby Achieve gene knockout. Detect the genotype of newborn rats by genotype identification and sequencing analysis. Use Real-time PCR, Western blot technology and immunohistochemistry to analyze the taurine transporter (taurine transporter) in rat brain tissue. ,TauT) mRNA expression and protein expression, and establish a Slc6a6 gene knockout rat model.

　　Results: There were 21 Slc6a6 knockout homozygotes (TauT-/-), 54 heterozygotes (TauT+/-), 27 negative (TauT+/+), and the homozygous rate of F3 generation was about 20.59%. It conforms to Mendelian genetic law. The mRNA level of the brain tissue of the Slc6a6 knockout homozygous rats is basically not expressed, and the TauT protein expression level is significantly lower than that of the littermate negative rats.

　　Conclusion: This study used the CRISPR/Cas9 system to target the Slc6a6 gene and successfully constructed a Slc6a6 gene knockout rat model.