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Fig. S1. A 361 bp core sequence is required for activity of the Tbx5 regulatory element constructs m5-10 and m5-5. (A) Sequence alignment between the m5-10 construct (Seq_1, lowercase) and the m5-10_del361bp construct (Seq_2, capital letters). The arrows indicate the start of the following constructs: m5-10, blue arrow; m5-5, blue arrow at nucleotide 5104; ΔRV, green arrow at nucleotide 5947; m5-5#3, purple arrow at nucleotide 9852; intron2, blue arrow at nucleotide 10044; intron2 MfeI, orange arrow at nucleotide 10698. These constructs span to the end of the sequence (position 11560). The 0.8RV construct corresponds to the sequence between red brackets (from nucleotide 5155 to 5946). The conserved sequence between chicken and mammals within this construct is highlighted in pink. The intron2 MscI construct contains the sequence between the intron2 arrow (forward, position 10044) and the intron2 MscI arrow (reverse, position 10605). The MscI(a) and MscI(b) segments are underlined in blue and red, respectively. The predicted Hox binding sites present in the 361 bp MscI(b) region are marked by grey rectangles and their position in the construct named #1 to #6. (B) Deletion of the 361 bp element in the background of the m5-5 construct (m5-5 Δint2core) results in a lack of reporter-driven β-gal expression in the developing forelimbs (0/7 transgenic embryos). The arrow points to a few cells with β-gal activity in the region of the heart, presumably an integration effect of the transgene insertion. (C) Similarly, deletion of the 361 bp element in the m5-10 construct (m5-10 Δint2core) result in loss of reporter activity (0/5 transgenic embryos).
