Figure 1.

Analysis of targetedStim1mutagenesis in DAT^Cremice and POMC neurons from Pomc^Cremice. A, schematic of a sagittal section showing the viral injections of AAV1-FLEX-SaCas9-U6-sg Stim1 and control virus into the VTA of adult male DAT^Cre mice. B, design of AAV1-FLEX-SaCas9-U6-sgStim1; the PAM is underlined. C, top 10 mutations at the cut site (black arrow) with the percent of total reads for which they occur on the left. Base changes: bolded. Insertions: underlined. Deletions: marked with a “-” (dash). D, frequency distribution of insertions and deletions in Stim1 from GFP⁺ nuclei. E, percent of wild-type, deletions, insertions, and base changes as percent of total reads for Stim1 in GFP⁺. F, schematic of a coronal section showing the bilateral viral injections in the ARH with AAV1-DIO-YFP. G, photomicrograph showing coronal section confirming targeted bilateral injections of AAV1-FLEX-SaCas9-U6-sgStim1 and control virus into the arcuate of adult male Pomc^Cre mice. Scale, 200 μM. H, quantitative PCR assay measuring Stim1 and Stim2 in POMC neuronal pools (n = 3 animals, 10 cells per pool, 5 pools/animal) from Pomc-EGFP male control mice. Bar graphs represent mean ± SEM (Unpaired t-test for the left, t₍₄₎ = 5.092, ∗∗p = 0.0070). n = animal number. I, quantitative PCR assay measuring Stim1 in POMC neuronal pools (n = 4 animals, 10 cells per pool, 4 pools/animal) using STIM1 (135 bp) primers that span the sgRNA and PAM from Pomc^Cre control and Stim1^Mut male mice. Bar graphs represent mean ± SEM (Unpaired t-test, t₍₆₎ = 5.275, ∗∗p = 0.0019).