Extended Data Fig. 6: Generation of mice deficient for Adcy3-at.
From: Cold-induced expression of a truncated adenylyl cyclase 3 acts as rheostat to brown fat function
Generation of transgenic mice deficient for Adcy3-at at exon 1 and TSS using CRISPR/CAS9 gene. a, Illustration of CRISPR-Cas9 mediated excision of Adcy3-at specific Exon 2b, including Adcy3-at TSS (light blue), yielding Adcy3-at specific knockout (Adcy3∆AT) mice. b, USCS genome browser track depicting mRNA (grey) and H3K4me3 signals (magenta) in the murine Adcy3 locus from chow diet fed, male C57BL/6N mice either housed at 22 °C (light grey) and exposed to 5 °C for 24 h (dark grey) and from HFD-fed, male C57BL/6N mice housed at 22 °C (light orange) and exposed to 5 °C for 24 h (dark orange) as determined by RNA-Seq and H3 K4me3 ChiP-Seq. Location and sequence of sgRNAs used for CRISPR/Cas9 mediated 978-nt deletion of Adcy3-at TSS are indicated. c, Southern blot of a control (C57BL/6N wildtype) and from Adcy3∆AT embryonic stem cell (ESC) genomic DNA. The asterisk depicts a heterozygous Adcy3∆AT/wild-type ESC clone. d, PCR from genomic DNA isolated from wild-type and Adcy3∆AT ESC genomic DNA. e,f, Relative expression of Adcy3-at (e) and Adcy3 (f) as determined by qPCR analysis in BAT, iWAT, and eWAT from chow diet fed, male LoxP (n = 4 for BAT and iWAT, n = 2 for eWAT) and Adcy3∆AT mice (n = 4) housed at 22 °C. Bar graphs represent mean + SEM with all data points plotted. An unpaired, two-tailed, and non-parametric Mann-Whitney test was performed to assess statistical significance. P values are indicated within the panel. Created with BioRender.com.
