Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA237021

Local Sample ID:	Ctrl_2_HC_Neg
Subject ID:	SU002450
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Treatment:

Treatment ID:	TR002462
Treatment Summary:	We used the inducible Cre/loxP technology to generate mice in which UCP2 was selectively ablated in POMC neurons (Ucp2PomcKO mice). First, mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) in cells expressing POMC (Pomc-CreERT2) were crossed with Rosa26-lox-stop-lox-tdTomato (Ai14; cre-recombinase-dependent expression) mice (Ai14 reporter mice; stock #007914; The Jackson Laboratory) to label POMC-expressing cells. Pomc-CreERT2; Rosa26-lox-stop-lox-tdTomato (Pomc-CreERT2; tdTomato) mice have POMC-expressing cells with the expression of tdTomato by tamoxifen administration. No observation of POMC-tdTomato expression was found in the absence of tamoxifen administration, indicating that recombination was strictly dependent upon tamoxifen-induced Cre recombinase activation. The mice with Pomc-CreERT2; tdTomato were then crossed with mice harboring conditional alleles Ucp2 floxed (Ucp2fl/fl; B6;129S-Ucp2tm2.1Lowl/J, Stock# 022394; The Jackson Laboratory) to generated mice with inducible deletion of Ucp2 specifically in POMC neurons (Ucp2PomcKO mice). All animal experiments were conducted with the mice expressing POMC-CreERT2. As control groups, Ucp2fl/fl; Pomc-CreERT2 mice were injected with tamoxifen, and Ucp2fl/fl; Pomc-CreERT2; tdTomato were mice injected with corn oil (vehicle), and Ucp2fl/fl; Pomc-CreERT2; tdTomato mice were injected intraperitoneally (IP) with tamoxifen (0.1 mg/g BW for 5 consecutive days) starting at 5 weeks of age to induce mature-onset deletion of Ucp2 in POMC neurons of Ucp2PomcKO mice. However, because there were no differences between these two control groups, most of the experiments were performed using Ucp2+/+; Pomc-CreERT2; tdTomato mice injected with tamoxifen (to label POMC neurons with tdTomato expression) as a control group, unless otherwise stated. Body composition was measured in vivo by MRI (EchoMRI; Echo Medical Systems, Houston, TX) monthly at 10:00 AM. We performed transcriptomic profiling by using a ribosomal tagging strategy to analyze POMC neuron-specific mRNA expression in vivo. We crossed Pomc-CreERT2 mice30 with Rpl22 floxed (RiboTag, Stock# 029977, The Jackson Laboratories) mice to generate Pomc-CreERT2; RiboTag mice, expressing a hemagglutinin A (HA)-tagged ribosomal protein in the POMC neurons upon tamoxifen injection.

Treatment ID:

TR002462

Treatment Summary:

We used the inducible Cre/loxP technology to generate mice in which UCP2 was selectively ablated in POMC neurons (Ucp2PomcKO mice). First, mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) in cells expressing POMC (Pomc-CreERT2) were crossed with Rosa26-lox-stop-lox-tdTomato (Ai14; cre-recombinase-dependent expression) mice (Ai14 reporter mice; stock #007914; The Jackson Laboratory) to label POMC-expressing cells. Pomc-CreERT2; Rosa26-lox-stop-lox-tdTomato (Pomc-CreERT2; tdTomato) mice have POMC-expressing cells with the expression of tdTomato by tamoxifen administration. No observation of POMC-tdTomato expression was found in the absence of tamoxifen administration, indicating that recombination was strictly dependent upon tamoxifen-induced Cre recombinase activation. The mice with Pomc-CreERT2; tdTomato were then crossed with mice harboring conditional alleles Ucp2 floxed (Ucp2fl/fl; B6;129S-Ucp2tm2.1Lowl/J, Stock# 022394; The Jackson Laboratory) to generated mice with inducible deletion of Ucp2 specifically in POMC neurons (Ucp2PomcKO mice). All animal experiments were conducted with the mice expressing POMC-CreERT2. As control groups, Ucp2fl/fl; Pomc-CreERT2 mice were injected with tamoxifen, and Ucp2fl/fl; Pomc-CreERT2; tdTomato were mice injected with corn oil (vehicle), and Ucp2fl/fl; Pomc-CreERT2; tdTomato mice were injected intraperitoneally (IP) with tamoxifen (0.1 mg/g BW for 5 consecutive days) starting at 5 weeks of age to induce mature-onset deletion of Ucp2 in POMC neurons of Ucp2PomcKO mice. However, because there were no differences between these two control groups, most of the experiments were performed using Ucp2+/+; Pomc-CreERT2; tdTomato mice injected with tamoxifen (to label POMC neurons with tdTomato expression) as a control group, unless otherwise stated. Body composition was measured in vivo by MRI (EchoMRI; Echo Medical Systems, Houston, TX) monthly at 10:00 AM. We performed transcriptomic profiling by using a ribosomal tagging strategy to analyze POMC neuron-specific mRNA expression in vivo. We crossed Pomc-CreERT2 mice30 with Rpl22 floxed (RiboTag, Stock# 029977, The Jackson Laboratories) mice to generate Pomc-CreERT2; RiboTag mice, expressing a hemagglutinin A (HA)-tagged ribosomal protein in the POMC neurons upon tamoxifen injection.