Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome
J. Clin. Invest. Michael Brunner, et al. 118:2246 doi:10.1172/JCI33578 [Go to this article.]

Figure 1
Transgenic constructs and expression studies. (A) Schematic drawings of the mutations (top) in KvLQT1 (left) and HERG (right) polypeptides and transgenic constructs (bottom). (B) PCR of genomic DNA of the founders: + and – denote positive control (constructs) and negative control. Numbers correspond to animals. Rabbits 2 (for KvLQT1) and 33 (HERG) tested positive. (C) Southern blot analyses of genomic DNA. Numbers in brackets indicate rabbit numbers. The sizes of the inserts incorporated into the rabbit genome (right panel) were identical to those of the plasmids (left panel). (D) Left panel shows Western blots of membrane lysates of CHO cells transfected with empty vector (control), KvLQT1, KvLQT1-Y315S, HERG, and HERG-G628S, and heart lysates from LMC, LQT1, and LQT2. Right panels show IP with anti-FLAG antibody of LMC, LQT1 (KvLQT1-Y315S), and LQT2 (HERG-G628S) crude heart lysates probed with anti-HERG and anti-KvLQT1 antibodies, respectively. The apparent molecular weight is 75 kDa for KvLQT1 and 135 kDa and 155 kDa for HERG. (E) Crude heart membranes were prepared from sections (LV and septum [S]) of LMC, LQT1, and LQT2. 200 μg samples were immunoblotted with polyclonal HERG antibody (top) and monoclonal KvLQT1 antibody (bottom). Crude membranes from CHO cells transfected with HERG and KvLQT1 cDNA served as positive controls. Anti-HERG antibody reacted with a 155-kDa polypeptide representing the endogenous RERG expression in LMC and LQT1 hearts. The anti-KVLQT1 mAb detected a 75-kDa band representing the endogenous rabbit KvLQT1 channel polypeptides in LMC and LQT2 hearts. Upper right panel shows crude membranes from LQT2 or LQT1 rabbit hearts, which were reacted with either anti-FLAG mAb (right lane) or mouse IgG (left lane). Anti-HERG antibody detected a 155-kDa polypeptide, while IgG failed to precipitate this peptide. Lower right panel shows anti-KvLQT1 mAb, which reacted with a 75-kDa band while IgG failed to precipitate it.