2022, Vol. 26 ›› Issue (32): 5179-5184

Construction of bromodomain-containing protein 4 gene knockout mice by CRISPR/Cas9 combined with Cre-loxP technology

Wang Xiangyu1, Zhu Ruizhi1, Zhao Zhiping1, 2, Zhang Yuda1, Zhang Yongtao3, Wang Changyao3   

Abstract: BACKGROUND: Currently, the combined use of CRISPR/Cas9 and CRE-LOXP to prepare bromodomain-containing protein 4 (BRD4) gene knockout mice is very rare.
OBJECTIVE: To construct a BRD4 gene knockout mouse model by using CRISPR/Cas9 technology combined with Cre-loxP technology that can be used to knock out the BRD4 gene fragment in the mouse genome.
METHODS: According to the exon sequence of BRD4 gene, a gRNA was designed and synthesized. After gRNA was transcribed in vitro, Cas9 mRNA and plasmid containing loxp site were injected into fertilized oocytes. Cas9 cut the target fragment by recognizing the leading strand of the gRNA, and then loxp was inserted into the cleavage site. After breeding with Cre, the Cre enzyme cut the loxp site to achieve the final specific deletion effect. The fertilized oocytes were transplanted into C57BL/6N recipient female mice to obtain progeny mice. The offspring mice were sequenced to identify their genotypes. The BRD4-loxP +/- (F0 generation) mice with loxp loci being successfully introduced was bred with wild-type C57BL/6N mice, and the stably inherited mice BRD4-loxP +/- (F1 generation) were selected. Some of the BRD4-loxP +/- mice interbred with each other and some interbred with CAGGcre-ERTM mice. BRD4-loxP +/+ mice were co-expressed with BRD4-loxP+/- and CAGGcre-ERTM mice (F2 generation). The F2 generation mice were bred to obtain homozygous BRD4-loxP +/+ and CAGGcre-ERTM mice. BRD4 gene knockout was induced by intraperitoneal injection of tamoxifen (75 mg/kg, dissolved in corn oil) in 6-8 weeks homozygous mice for 7 consecutive days. DNA was extracted from the tail fragment of the knockout mice using adsorption column method. The expression of BRD4 gene fragment in mouse tail tissue was detected by agarose gel electrophoresis.
RESULTS AND CONCLUSION: Screening using PCR revealed that mice 7, 8, 9, 10, 12, 14, 16, 19, 20, 21, 25, 42, 43, and 47 of F1 generation were identified as heterozygous mice that were successfully inserted with two alleles of loxP fragment. The F3 homozygotes bred from F2 generation were induced with tamoxifen, and PCR gel electrophoresis verified that the BRD4 gene fragment was successfully knocked out in the homozygous mice. We therefore successfully constructed BRD4 gene knockout mice using CRISPR/Cas9 technology combined with CRE-LOXP technology in this study.
Key words: CRISR/Cas9, Cre-loxP, BRD4, gene knockout, mouse, tamoxifen