Generation and identification of carbohydrate response element-binding protein conditional gene knockout mouse model / 第二军医大学学报

Objective To establish a conditional knockout mouse model of transcription factor carbohydrate response element-binding protein (ChREBP), so as to provide a technical approach for exploring the biological function of ChREBP in vivo. Methods and results Using Cre/loxP gene targeting strategy, we constructed the gene targeting vector and franked the 8th exon of ChREBP gene with loxP site in embryonic stem cells (ES cells) via homologous recombination. We microinjected the recombined ES clones into blastocysts and implanted into the uterus of pseudopregnant mice to obtain the chimeric mice and ChREBPflox/+ mice. By crossbreeding ChREBPflox/+ mice with Alb-Cre mice, we generated liver-specific ChREBP knockout mice. Conclusion The generation of ChREBP conditional knockout mouse model provides an important means to reveal the physiological function and pathological significance of ChREBP in different tissues.

Transcription factor ChREBP-α enhances lipogenesis of primary hepatocytes / 第二军医大学学报

Objective To construct recombinant adenovirus expressing mouse ChREBP-α, and examine the effect of ChREBP-α overexpression on lipid synthesis in mouse primary hepatocytes. Methods The mouse ChREBP-α cDNA was subcloned into pShuttle-CMV vector, and the product was transformed into E. coli strain BJ5183 for homologous recombination with pAdEasy-1.The resultant recombinant vector was transfected into 293A cells for viral package. Mouse primary hepatocytes were infected with adenoviruses Ad-ChREBP-α, and gene expression was analyzed at mRNA and protein levels by real-time PCR and Western blotting analsysis. The expression levels of ChREBP target gene LPK were measured at mRNA level by real-time PCR, and the fatty acid synthesis rate was determined by [14C]-acetate incorporation. Results We successfully constructed recombinant adenoviruses expressing mouse ChREBP-α. Adenovirus-mediated overexpression of ChREBP-α markedly increased LPK mRNA expression and fatty acid synthesis in primary hepatocytes. Conclusion We have successfully constructed recombinant adenoviruses expressing mouse ChREBP-α, which is biologically active and can overexpress ChREBP-α in primary hepatocytes. Overexpression of ChREBP-α can enhance lipid synthesis.