To confirm BptfXG023 and BptfΔexon2 expression and an out of frame Bptf mRNA in the BptfΔexon2 line we designed primers with homology to the 3′ end and exons 1 through 8 of the Bptf mRNA. Total RNA from wild type and BptfΔexon2 homozygous embryos was converted to cDNA using superscript II according to manufacturer's procedures. Bptf expression was estimated by first normalizing cDNA to equal concentration with GAPDH amplification. Following GAPDH normalization 3′Bptf was amplified. Both GAPDH and Bptf were amplified in the linear range as follows. Reactions were heated for 10 min at 94°C, then cycled for 22 cycles for GAPDH and 30 cycles for Bptf at 20 sec at 94°C, 20 sec at 60°C, and 30 sec at 72°C. PCR products were resolved by native PAGE. The exon 1–8 junction was amplified using primers P3 and JL15 in 25 µl volumes with 20 mM Tris-HCL (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.2 µM each primer, 1 Unit Taq Polymerase (Invitrogen) (see Table S3 for primer sequences). Reactions were heated for 3 min
