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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).
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