Generation and identification of knock-in mice.

<p>A) Construct and targeted genomic DNA before and after homologous recombination. The region of exon 7, which codes for the dipeptide sequence, the two YXXΦ motifs and the histidine-rich sequence (amino acid residues 441–529), was replaced by an in-frame GFP sequence. An FPL-flanked neo cassette was inserted downstream of the FgfrL1 gene to enable selection of positive clones by G418. This cassette was subsequently removed by mating with FLP-mice. Kinked arrows show the translation initiation sites of FgfrL1 and Neo, respectively. Arrowheads show the relative position of primers used for genotyping. The sketch is not drawn to scale. B) Genotyping of mutant mice by PCR. Samples from wild-type animals yielded a unique fragment of 504 bp, whereas samples from knock-in mice gave a unique fragment of 966 bp. A sample lacking any genomic DNA (negative control) and a DNA standard were included on the gel. C) Northern blots with samples from mutant mice. Total RNA from the tongue of wild-type, heterozygous and homozygous knock-in mice at E18.5 was separated on an agarose gel, transferred to a Nylon membrane and hybridized with radiolabeled probes for FgfrL1 and GFP, respectively. The 28S RNA stained with ethidium bromide was included as a loading control.