B6 cg tg alb cre 21mgn j

C57Bl6/J mice were from the TSRI breeding colony. Bmal^L/L mice harboring loxP sites surrounding exon 8 of the Bmal1 gene (stock #007668, (B6.129S4(Cg)-Arntl^tm1Weit/J) and Albumin-Cre mice (stock #003574, B6.Cg-Tg(Alb-cre)21Mgn/J) were purchased from Jackson Laboratories (Bar Harbor, ME) and were bred to obtain Bmal1^L/L (control) and Bmal1^L/L;Albumin-Cre^Tg/+ (L-Bmal1^−/−) littermates. Standard genotyping protocols with primers from the Jackson Laboratories strain information pages were used. Mice were entrained to a 12-h light/12-h dark cycle for at least 2 weeks prior to experiments and all experiments were performed at 11–14 weeks of age. All animal care and treatments were in accordance with The Scripps Research Institute guidelines for the care and use of animals and were approved by the TSRI Institutional Animal Care and Use Committee (IACUC).

Generation of Acly^f/f mice on a C57Bl6/J background was previously described^{10 (link)}. To generate hepatocyte-specific Acly knockouts, Acly^f/f mice were crossed to albumin-Cre transgenic mice (B6.Cg-Tg(Alb-Cre)21Mgn/J, Jackson Laboratory)³⁷ .

A CRISPR/Cas9 gene-editing strategy was adopted to produce Mrs2 knock out (KO) mouse embryonic fibroblasts (MEFs). Briefly, candidate sgRNAs were selected based on a CRISPR/Cas9 nickase design platform, which substantially reduces off-target effects by ~50–1500 fold. The selected paired gRNAs targeting exon 8 of Mrs2 (the Cas9 nickase mRNA and single-stranded oligodeoxynucleotide (ssODN)) for HDR-mediated gene KO were transfected into MEFs. Knockout was confirmed by genotyping by PCR followed by restriction fragment analysis with BamH1 (Figure 4B). The Mrs2 targeted allele was cleaved yielding two fragments (388 and 368 bp), whereas the wild type (WT) allele was not. After validation of sgRNAs and ssODN in vitro, gRNA1, gRNA2, ssODN containing the BamHI site along with Cas9 protein was microinjected into single-celled embryo and transferred to surrogate mother. Details of the oligo’s sequence used for the Mrs2 KO are provided in Figure 4B. The chimeras were bred with C57BL/6J (The Jackson Laboratory, USA) mice over 10 generations, and both sexes were used for in vitro and in vivo studies. Hepatocyte-specific Mcu knockouts (MCU^Δhep) were generated by crossing MCU^fl/fl mice (Tomar et al., 2019 (link)) with hepatocyte-specific Cre recombinase transgenic mice (B6.Cg-Tg(Alb-cre)21Mgn/J, The Jackson Laboratory, USA).

Hepatocyte-specific Mcu knockouts
(MCU^Δhep) were generated by crossing
MCU^fl/fl mice (Luongo et al.,
2015) with hepatocyte-specific Cre recombinase transgenic mice
(B6.Cg-Tg(Alb-cre)21Mgn/J, The Jackson Laboratory, USA). Hepatic AMPK-KO
(AMPK^Δhep) mice were generated by retro orbital
injection of the 1×10¹¹ viral particles of adenovirus
associated virus 8-Cre (AAV8-Cre under the control of the TGB promoter) in
the AMPKα1/α2^fl/fl mice which have the Lox-P site
on both AMPKα1 and AMPKα2 loci. Mice were allowed to rest for
2 weeks before the experiment. To generate MCU-C96A knock in mice, gRNA1,
gRNA2, ssODN containing BssHII site along with Cas9 protein was
microinjected in single-celled embryo and transferred to surrogate mother.
Details of oligo’s sequence used for C96A-KI are given in Figure S5B. The
C96A-KI is confirmed by restriction fragment analysis using BssHII
restriction enzyme. All mice were grouped according to same sex and age for
all the experiments. For studies utilizing primary hepatocytes in
vitro, both male and female mice were used. However, male mice
were used for all in vivo experiments. All animal
experiments were approved by Temple University’s IACUC and followed
AAALAC guidelines.

Il22ra1^fl/fl mice on a C57BL/6 background were designed in our laboratory and made at Ozgene. LoxP sites were engineered to flank exon 3. To determine functional recombination, Il22ra1^fl/fl mice were injected in the tail vein with an E1-deleted adenovirus expressing Cre recombinase and liver DNA was extracted and subjected to PCR using primers spanning exon 3. To determine functional deletion of IL-22RA1 specifically in the liver of Il22ra1^fl/fl mice, we crossed this line to B6.Cg-Tg(Alb-cre)21Mgn/J or Albumin-Cre mice (obtained from The Jackson Laboratory), which confirmed cre-mediated DNA recombination by allele specific PCR. Ccsp-Cre mice were obtained as a kind gift from Dr. Thomas Mariani and described before(16 (link)). Il22^−/− mice were obtained from The Taconic Farms.

Targeted C57BL/6J ES cells with Abcb10 containing loxP sites flanking exons 2–3 and a neomycin cassette flanked by Frt sites were generated by Genoway. Targeted ES cells were injected and implanted in C57BL/6J-Tyrc-2J/J (albino) females at the Boston University Mouse Transgenic Core, directed by Gregory L. Martin and Katya Ravid. Seven male chimeras containing floxed ABCB10 were identified by coat color and bred with C57BL/6J FLP recombinase females from Jackson (B6.Cg-Tg(ACTFLPe)9205Dym/J) to excise the neomycin cassette. The offspring were bred with wild type C57BL/6J, to obtain Abcb10^wt/flox mice without FLP recombinase. Abcb10^wt/flox mice were paired with B6.Cg-Tg(Alb-cre)21Mgn/J purchased from Jackson, to generate ABCB10 liver-specific KO mice. Groups analyzed were offspring littermates from breeding pairs between Abcb10^flox/flox; Alb-Cre^−/− with Abcb10^wt/flox; Alb-Cre^+/−. Wild type mice (WT) are Abcb10^flox/flox and Abcb10^wt/flox mice (no Cre) and ABCB10-LKO are Abcb10^flox/flox; Alb-Cre^+/−.