Figure 1.

Generation of conditional Ube2i gene deletion mice. (A)Ube2i^fl/fl mice were generated using CRISPR/Cas9 gene editing. Exons 3 and 4 of the Ube2i (Ubc9) gene were targeted by sgRNAs designed complementary to intronic sequences flanking the exons, then loxP sequences were introduced by DNA donor oligonucleotides. LoxP sites were inserted before exon 3 and after exon 4 (black arrows). Primers detect the 5’ (P1, P2) and 3’ (P3, P4) loxP sequences. (B) PCR analyses of floxed alleles at the targeted loci in genomic DNA extracted from ear clips of wild-type (+/+), fl/+, and fl/fl mice. PCR products were run on agarose gels with expected band sizes for P1–P2: wild-type (+) 319 bp and loxP allele (fl) 353 bp and P3–P4: wild-type (+) 427 bp and loxP allele (fl) 461 bp. The image shows a wild-type control #1107 +/+, founder heterozygous mouse #974 fl/+, and homozygous F2 offspring #1091 fl/fl. (C) Western blot analysis of UBE2I expression in inguinal WAT (iWAT) derived fibroblasts from Ube2i^fl/fl mice after transduction with adenoviral GFP or Cre. To determine cell autonomous effects of Ube2i deletion in adipocytes, tamoxifen inducible Cre-expressing mice (CAG-Cre) were crossed with Ube2i^fl/fl mice. iWAT SVF cells were isolated from CAG-Cre;Ube2i^fl/fl and Ube2i^fl/fl control mice. All cells were treated with tamoxifen to induce Cre recombination followed by adipocyte differentiation (diff) for eight days. (D) Western blot analysis and (E) relative gene expression by qPCR were performed to assess Ube2i depletion, adipocyte maturation, and beige fat cell markers. Gray = Ube2i^fl/fl, yellow = CAG-Cre;Ube2i^fl/fl. Data are presented as mean +/− SEM, ∗p < 0.05. (F) Brightfield and fluorescent images of differentiated Ube2i^fl/fl and CAG-Cre;Ube2i^fl/fl cells stained for lipids (green, LipidTOX), perilipin (red), and nuclei (blue, DAPI). Scale bars 50 μm. (G) Whole cell lysates from differentiated cells were subjected to Western blot analysis of cleaved (p41/43, p18) and uncleaved Caspase-8. (H) Strategy for generating adipocyte-specific Ube2i gene deletion. Ube2i^fl/fl mice were bred with Adipoq-Cre mice to generate adipocyte-specific Ube2i knockout (Ube2i^a-KO) and Ube2i^fl/fl (control) mice. (I) To validate gene deletion of Ube2i, genomic DNA was extracted from Ube2i^a-KO and Ube2i^fl/fl gonadal WAT (gWAT) samples and PCR products were run on an agarose gel to detect the 5’ (P1, P2 primers) and 3’ (P3, P4 primers) loxP sequences, as well as a product that spans exons 3–4 (P1+P2+P4; 1597 bp, green arrow) or the deletion product (509 bp, red arrow). (J) Western blots of whole tissue lysates from iWAT and BAT of seven-day-old mice were probed for UBE2I and adiponectin (ADIPOQ), and HSP90 served as the invariant loading control.