We bred all mice in our colony in the Unit for Laboratory Animal Medicine at the University of Michigan. All animals and procedures used were in accordance with the guidelines and with the approval of the University Committee on the Use and Care of Animals. We provided all animals ad libitum access to food and water. We purchased male C57Bl/6J animals for breeding studies and Gt(ROSA)26Sortm1(EYFP)Cos (ROSAEYFP) mice from Jackson Labs. We produced PomcEYFP and AgrpEYFP animals by crossing PomcCre and AgrpCre animals5 (link),36 (link) onto the ROSAEYFP background.
To generate Nos1cre, PCR amplification produced a 6 kb fragment containing the mouse genomic Nos1 sequence centered on the STOP codon in the final (3’) exon for insertion into pCR2.1. PCR mutagenesis created an AscI site 60 bp 3’ to the STOP codon, for the introduction of the IRES-Cre-Frt-Neo-Frt sequences to generate pCRNos1-IRES-Cre-Frt-Neo-Frt. NotI/NheI digestion excised the entire insert for subcloning into NotI/XbaI-cut pPNT backbone to generate pPNT-Nos1-IRES-Cre for targeting. NotI digestion linearized the vector for electroporation into R1 ES cells. We used Taqman-based qPCR screening to initially identify correctly targeted clones37 (link), followed by Southern blotting for final confirmation. We injected correctly targeted ES cells into blastocysts to generate chimeras, which we bred to C57Bl/6 animals to establish germline transmission.
We bred Nos1cre mice with ROSAEGFP mice to generate Nos1cre;ROSAEGFP (Nos1EGFP) animals for the analysis of Nos1cre expression. We also bred Nos1cre animals with Leprfl/fl mice. Due to the periodic expression of Nos1cre during gametogenesis, we bred Nos1cre;leprΔ/+ to Leprfl/fl mice in order to obtain littermate Nos1cre;LeprΔ/fl (LeprNOS1KO), Nos1cre; LeprΔ/+ and LeprΔ/fl (control) and Nos1cre;LeprΔ/Δ (LeprKO) animals for study. We genotyped the offspring by PCR.
To generate Nos1cre, PCR amplification produced a 6 kb fragment containing the mouse genomic Nos1 sequence centered on the STOP codon in the final (3’) exon for insertion into pCR2.1. PCR mutagenesis created an AscI site 60 bp 3’ to the STOP codon, for the introduction of the IRES-Cre-Frt-Neo-Frt sequences to generate pCRNos1-IRES-Cre-Frt-Neo-Frt. NotI/NheI digestion excised the entire insert for subcloning into NotI/XbaI-cut pPNT backbone to generate pPNT-Nos1-IRES-Cre for targeting. NotI digestion linearized the vector for electroporation into R1 ES cells. We used Taqman-based qPCR screening to initially identify correctly targeted clones37 (link), followed by Southern blotting for final confirmation. We injected correctly targeted ES cells into blastocysts to generate chimeras, which we bred to C57Bl/6 animals to establish germline transmission.
We bred Nos1cre mice with ROSAEGFP mice to generate Nos1cre;ROSAEGFP (Nos1EGFP) animals for the analysis of Nos1cre expression. We also bred Nos1cre animals with Leprfl/fl mice. Due to the periodic expression of Nos1cre during gametogenesis, we bred Nos1cre;leprΔ/+ to Leprfl/fl mice in order to obtain littermate Nos1cre;LeprΔ/fl (LeprNOS1KO), Nos1cre; LeprΔ/+ and LeprΔ/fl (control) and Nos1cre;LeprΔ/Δ (LeprKO) animals for study. We genotyped the offspring by PCR.
