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For determining the Pomc copy number, 20 ng of genomic DNA extracted via standard isopropanol precipitation from tail biopsies was used. Selected gene segments were amplified using TaqMan Universal PCR-Master Mix (Thermo Fisher Scientific, 4305719) according to the manufacturer’s instructions. Non-exon-spanning primers (P1: 5′-GCGACAGGGACCAAACGG-3′, P2: 5′-AGACACCCTTACCTGTCGC-3′) and probes (5′-FAM-TCAGTGGCCTCTCTTAGTCACTGC-TAMRA-3′) were designed for amplification of Pomc exon 1, while the gene expression assay for Socs3 (Thermo Fisher Scientific, Mm00545913_s1, 4331182) was purchased. The expression of Pomc exon 1 was normalized to Socs3 mRNA. Results were calculated by the ΔCt comparative method (as 2−ΔΔCt), in which fold changes were calculated by defining wild-type animals with two Pomc gene copy numbers.

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