Generation of Ucp1-ires-Cre knock-in mice to enhance specificity and efficiency of gene targeting in brown adipose tissue

Abstract

Brown adipose tissue (BAT) secretes cytokines that influence the function of other tissues. Given the widespread distribution of brown fat depots, we generated BAT-lacking (ΔBAT) mouse models by specifically eliminating brown adipocytes using the Cre-loxP system combined with a floxed-stop diphtheria toxin A (DTA) cassette. Uncoupling protein 1 (UCP1) is essential for BAT thermogenesis and exhibits a highly restricted expression pattern, so it was chosen to direct BAT-specific Cre recombinase expression. We used CRISPR-Cas9 to insert an ires-Cre sequence downstream of the UCP1 stop codon, developing the novel knock-in line, Ucp1-Cre^YH. Ucp1-Cre^YH and transgenic line TgUcp1-Cre^Evdr mouse (Ucp1-Cre^Evdr) were crossed with Ai14-tdTomato and floxed-CPEB2 mice to assess Cre specificity and efficiency. ΔBAT mice were then generated by crossing each Cre line with floxed-stop DTA mice, followed by assessments of locomotor activity, body weight, and glucose tolerance. Although both Cre lines showed cold-enhanced expression, Ucp1-Cre^Evdr exhibited considerably lower Cre levels in BAT compared with Ucp1-Cre^YH mice, leading to inefficient ablation of some floxed alleles, such as Cpeb2. Moreover, Ucp1-Cre^Evdr mice displayed nonspecific Cre expression, whereas neither line showed evidence of substantial autonomous Cre activity in BAT-resident macrophages. Consequently, ΔBAT^Evdr mice experienced off-target neuronal ablation, resulting in hyperactive locomotion and reduced body weight. Although ΔBAT^YH mice showed normal locomotor activity and body weight, they had a modest weight gain and altered glucose homeostasis only after high-fat-diet feeding. In conclusion, novel Ucp1-Cre knock-in mouse showed specificity and efficiency for gene manipulation in brown adipocytes, highlighting its application in generating BAT-specific knockout and BAT-depleted mouse models.NEW & NOTEWORTHY A novel Ucp1-CreYH knock-in mouse exhibits improved efficiency and specificity of Cre recombinase activity in brown adipose tissue (BAT) compared with the widely used Ucp1-CreEvdr line, which showed nonspecific Cre activity in many organs. A BAT-deprived mouse model, generated with this Cre line, showed normal locomotion and body weight, but altered body weight and glucose homeostasis after high-fat feeding, validating its use for BAT-specific gene manipulation.

Keywords: BAT-depleted mice; Ucp1; body weight; brown adipose tissue; glucose homeostasis.