DEV02018 Supplementary Material

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• Supplemental Figure 1 - Fig. S1. Evaluation of TgCAGG-CreER^TM-induced recombination. (A) Alternative PCR strategy to evaluate Tbx1^flox recombination. The primer pair (arrows) amplifies two large fragments from the Tbx1^flox and wild-type (Tbx1⁺) alleles, 1100bp and 1000bp, respectively; and a small, 270 bp fragment from the Tbx1^DE5 allele. The large arrowheads flanking exon 5 (E5) in Tbx1^flox allele are loxP sites. Also shown are representative PCR results from genomic DNA extracted from individual Tbx1^flox/+ embryos (controls, Cre-) and TgCAGG-CreER^TM;Tbx1^flox/+ embryos exposed to TM in utero for the time indicated. (B) The strategy of reverse-transcription (RT)-PCR to detect residual Tbx1 mRNA in embryos after tamoxifen-induced, Cre-mediated recombination. The PCR primer pair (arrows) amplifies a 239 bp fragment from RNA transcribed from the Tbx1^flox and Tbx1⁺ alleles, and a 66bp fragment from RNA transcribed from the Tbx1^DE5 allele. Representative RT-PCR results using RNA extracted from individual embryos at the stages indicated are shown. All the embryos were exposed to TM in utero for 24 hours. Control, TgCAGG-CreER^TM; Tbx1^flox/+ (except for the one with the asterisk, which is Tbx1^flox/+); mutant, TgCAGG-CreER^TM; Tbx1^flox/-. b-Actin is used here as an internal control. (C) TgCAGG-CreER^TM; R26R embryos exposed to tamoxifen in utero for 24 hours and stained with X-gal. Examination of sections throughout the embryos revealed homogeneous staining of tissues of the pharyngeal apparatus. Scale bar: 1 mm.