Fig. 1. Incorporating the Tet system into the Hand1 locus to generate an overexpression model. (A) Wild-type Hand1 consists of two exons (boxes: non-coding regions indicated in white, coding region is shaded, bHLH domain is shown in black) separated by a 1.5 kb intron. (B) The targeted Hand1 locus contains the transactivator tTA (red) under the control of the endogenous Hand1 promoter. (C) The tet-responsive Tre2 (blue)-Hand1 cDNA (black) upstream of a ß-globin poly(A) (green) randomly integrated as a transgene. The primers PrA, PrB, PrC and PrD are as shown in A,B. (D) Southern blot analysis to confirm the targeting of the endogenous Hand1 locus for the driver and varying copy number founder lines for the responder. (i) BglII digest with the 5' probe illustrated in A. (ii) HindIII digest with the probe illustrated in C. (E) PCR genotyping of F1 following driver x responder matings (asterisk denotes compound driver/responder heterozygote). (F) Quantitative real-time PCR on RNA from compound heterozygote embryos at E9.5 reveals twice as much expression of Hand1 compared with control littermates containing only driver or responder. Bars represent mean±s.e.m. of three real-time qRT-PCR experiments on single founder overexpression and control embryos. co, control; ns, non-specific; oe, overexpression.