Figure 1: Selective Ablation of
2019-06-11T07:00:54Z (GMT) by
(A)Abcc9 encodes sulfonylurea receptor 2 (SUR2), the major SUR expressed in the myocardium. Shown is a schematic of Abcc9-produced transcripts encoding SUR2 full-length and SUR2-55KDa forms. Exon 5 is shown in red. (B) Shown is a depiction of full-length SUR2 containing 3 transmembrane domains (TMDs): TMD0, TMD1, and TMD2. SUR2-55KDa protein contains TMD0 and a portion of TMD2. The position of the protein domains encoded by exon 5 is shown with a red box. (C) Abcc9 gene targeting of exon 5 with LoxP sites (black triangles) flanking exon 5 (red). These mice, referred to as Fl Ex5 (floxed exon 5), were bred to mice carrying a transgene which expresses Cre recombinase under the control of the Myh6 promoter, MerCreMer (MCM), creating the cardiac Fl Ex 5 mouse model (19). (D) Tamoxifen dosing strategy for deletion of Abcc9 exon 5. (E) Polymerase chain reaction (PCR) of genomic deoxyribonucleic acid (DNA) isolated from tail clip or cardiomyocytes (CMs) from MCM Cre– and MCM Cre+ Fl Ex5 mice using a 3-primer strategy to detect the deletion of exon 5 (upper band) or floxed exon 5 (lower band). (F) Reverse transcriptase PCR (RT-PCR) from complementary DNA (cDNA) of tamoxifen-treated MCM Cre– and MCM Cre+ mice show the exon 5–including transcript (upper band) and the transcript generated from the exon 5 deletion (lower band). Products confirmed by Sanger sequencing. (G) Quantitative PCR analysis of cDNA from ventricular myocardium from tamoxifen-treated MCM Cre– and MCM Cre+ mice show reduced total Abcc9 transcript levels in Cre+ mice (n = 4, 3; p = 0.008). (H, I) Immunoblot analysis of hearts from tamoxifen-treated MCM Cre– and MCM Cre+ mice show reduction of full-length SUR2 protein (n = 8). *p < 0.001. (J) Quantitative PCR analysis of cDNA from isolated ventricular myocardium from tamoxifen-treated MCM Cre– and MCM Cre+ mice show equal transcript levels of Kcnj8 (n = 5, 3). gDNA = genomic DNA; Tg = transgenic; WT = wild-type.