Fig. 2. Tfr transgene construct and expression. (A) Schematic showing the Tfr construct. Segments derived from the mouse Pax6 gene are shown in reds and include the ectoderm enhancer (EE) and the P0 promoter. The promoter is encompassed in a fragment of approximately 1 kb 5' to the start-point of transcription (right-facing arrow). The Fgfr1IIIc cDNA is positioned downstream of the Pax6 gene elements. The various protein-coding domains within the cDNA are include the signal sequence (SS), three imunoglobulin-like domains (Ig1-3), the acidic domain (blue vertical bar), the CAM-homology domain (purple vertical bar) (Doherty and Walsh, 1996) and the transmembrane domain (dark green vertical bar). Included in the construct is the SV40 virus small t antigen intron and polyadenylation signals. (B-E) Whole-mount in situ hybridization on mouse embryos. (B) Control hybridization with an antisense probe to 
A-crystallin showing signal in the lens pit in an E10.5, wild-type embryo (1ba – first branchial arch). (C,D) Hybridization of an antisense SV40 probe to Tfr7 embryos at E10.5 (C) and E11.5 (D). Hybridization signal is apparent in the lens pit at E10.5 and the lens vesicle at E11.5. (E) Hybridization of an antisense SV40 probe to a Tfr3 embryo at E12.0 showing signal in the lens epithelium. The broken line in D,E outlines the optic cup. (F) RT-PCR transgene expression analysis on lens placode and optic vesicle (E9.5) from Tfr7 hemizygous mice. Bacteriophage 
X174 DNA digested with HaeIII is run as a size standard on both sides of the gel (X). The GAPDH cDNA-derived product appears at 420 bp (blue arrowhead). The PCR product derived from the transgene appears at 200 bp (red arrowhead). This analysis shows that reverse-transcriptase (RT) must be included if either PCR product is to be amplified and that the transgene-specific product can be amplified only from lens placode cDNA.
