Fig. 1. Generation and analysis of mutant BAC transgenes. (A) Schematic drawing of Fgfr1, harbouring three extracellular immunoglobulin (Ig)-like domains, with relative contributions of exons 6-9 indicated (dashed arrows). The grey region in the C-terminal half of the third Ig loop (III) shows the position of alternative splicing between the IIIb and IIIc isoforms, generated by the use of exons 8 or 9, respectively. Dashed lines expand and highlight the nucleotide sequence of the linker region where the Pro252Arg activating mutation (BAC16) and Leu257Leu silent mutation (BAC15) were introduced by base substitutions. Note that the modifications also introduce MspI-sensitive (BAC16) or PstI-sensitive (BAC15) sites (underlined). (B) FISH analysis, using Fgfr1 probes on metaphase chromosomes, shows integration of BAC transgene into chromosome 4 (arrows). This particular sample was derived from a 4C-transgene animal (see text) and, accordingly, the signal from the transgene is twice as intense as that emanating from the endogenous allele on chromosome 8 (arrowheads). (C) Reverse-transcriptase PCR (RT-PCR) products generated by primers from exons 6 and 8, exposed to MspI enzyme and resolved on a 3% agarose gel. Left and right lanes, products derived from wild-type and BAC16 mouse liver RNA, respectively. Note that the level of transgene expression (303 bp) is equal in intensity to that of the wild-type (343 bp). (D) Resolution of PCR products amplified from tail genomic DNA and digested with MspI. In the left lane (2C), the intensity of the mutant bands is equal to that of the wild type, whereas in the right lane, the mutant band is almost twice as bright. Measurements of these intensities using NIH-imaging software confirm a 2:2 ratio for the 2C, and greater ratios for the 4C mutants (values not shown).