Fig. 3. The neo-r cassette causes the production of a hybrid Gbx2 transcript and a reduction of wild-type Gbx2 transcripts in Gbx2^neo homozygotes. (A) Schematic representation of the Gbx2^neo allele and the hybrid Gbx2^neo transcript produced by aberrant splicing into and out of the neo-r cassette. Open arrows indicate the transcriptional direction of Gbx2 and neo-r. Arrowheads indicate the location of primers used for RT-PCR. Larger boxes indicate exons (hatched, non-coding; red, coding; HB, homeobox). Rectangles indicate loxP sites. Sequences surrounding the splice junctions are indicated (red, Gbx2; gray, neo-r). (B) RT-PCR amplifies both wild-type and hybrid Gbx2 transcripts only in Gbx2^neo homozygotes. (C) Whole-mount ISH demonstrates a qualitative reduction of Gbx2 expression at E7.75 and 12-13 ss. (D) Quantitative RT-PCR illustrating that wild-type levels of Gbx2 transcript are reduced to 6-10% of normal in Gbx2^neo homozygotes at 6-8 ss and 24 ss. y-axis, number of Gbx2 transcripts (log 10); x-axis, genotype (–rt, control samples without reverse transcription).