Fig. 1.

WT and mutant CA box β4 promoter transgenic constructs. (A) WT and mutant β4 promoter/lacZ transgene architecture. The β4/α3/α5 gene cluster is depicted as boxes with arrows depicting the direction of transcription. Below the clustered nAChR subunit genes is a schematic of the linearized construct used to generate the transgenic animals. MAR, matrix attachment region; NLS, nuclear localization sequence. Shown below this schematic are the nucleotide sequences of the WT and mutant CA boxes. Mutations made to the CA box in the mutant transgenic construct are shown in grey with asterisks over the mutated nucleotides. (B) β4 promoter/lacZ transcriptional activity in vitro. DNA from either the WT (black bars) or mutant (white bars) transgenic constructs was transfected into Neuro-2a (left) or SN17 cells (right) along with a luciferase construct in which the SV40 promoter drives expression of the firefly luciferase gene. β-Gal activity was normalized to luciferase activity in order to correct for differences in transfection efficiencies. The data shown here are an average of 3 independent experiments, error bars represent standard deviations of the means. Student t test indicated that CA box mutation significantly decreased the β-gal activity of the mutant transgenic construct in both cell lines, p<0.05.