Figure 1.
(A) Schematic diagram of a single Tet‐off autoregulated AAV expression system: pAAV‐Tetoffbidir‐Alb‐luc vector. Vector encodes luciferase or furin‐cleavable codon optimized modified human proinsulin gene under the control of Alb, the albumin promoter. ITR, inverted terminal repeat; luc, luciferase gene; polyA, SV40 fragment containing the early and late polyadenylation signals; tTA‐advanced, mutated tetracycline transactivator; TRE‐tetracycline response element that contains seven copies of tetracycline operator (TetO). (B) Constitutive versus regulatable luciferase expression by doxycycline. HepG2 cells were transfected with either a constitutive liver specific pAAV‐HLP‐Luc vector or pAAV‐Tetoffbidir‐Alb‐luc vector. The transfected cells were induced with 0, 0.5 and 1 μg/ml doxycycline for 24 hours. Luciferase activities were assayed using Nano‐Glo® dual‐luciferase® reporter (NanoDLR™) assay system (Promega). Readings from each well were normalized to signals from co‐transfected controls (pSV40‐NlucP). Data are reported as the mean ± SEM from triplicate wells in a single experiment