动物造模,基因敲除 z-bio 2022-03-04 548

　　Objective To construct a conditional knockout rat of taurine transporter in the central nervous system by combining CRISPR/Cas9 and Cre-loxP technology, and to conduct preliminary research on mitochondrial mtDNA and mitochondrial respiratory chain enzyme activity in brain tissue.

　　Methods Using CRISPR/Cas9 and Cre-loxP technology, heterozygous rats (TauTloxP/WT) containing loxP sites at both ends of TauT gene exon 5 were obtained. The obtained TauTloxP/WT rats were mated with Nestin-Cre rats, and finally neural-specific TauT knockout rats (TauTloxP/loxP/Cre+) were obtained after breeding and identification. Real-time PCR, Western blot and immunohistochemistry were used to detect gene and protein expression in TauTloxP / loxP / Cre+ rats, and HE staining was used to observe the morphology of brain tissue. /Ⅱ/Ⅲ/Ⅳ/Ⅴ) activity was detected.

　　Results Compared with wild-type rats, the expression of TauT gene and protein in the brain tissue of TauTloxP / loxP / Cre+ rats was significantly decreased, the TauT gene was successfully knocked out in the central nervous system, and the model was successfully constructed; HE staining showed that TauTloxP / loxP / Cre+ The number and density of brain cells in rats decreased, while the cell lesions in the brains of aged TauTloxP/loxP/Cre+ rats were more obvious. In addition, compared with wild-type rats, the activities of mitochondrial respiratory chain complex enzymes I, III, IV and V in the brain tissue of TauTloxP/loxP/Cre+ rats were significantly decreased, but the mtDNA copy number was significantly increased.

　　Conclusion Using CRISPR/Cas9 and Cre-loxP technology, the central nervous system TauT gene knockout rat model was successfully constructed, and the effect of this model on brain tissue and mitochondrial respiratory chain enzymes and mtDNA was preliminarily verified. TauT provides a new model platform for the molecular mechanisms of brain tissue.