Exons 14 and 15 of the mouse Ret gene are flanked by loxP sites in this conditional mutant strain. Cre-mediated excision of the floxed region results in a knockout allele. A V805A mutation in exon 15 is functionally silent, but sensitizes the protein to chemical inhibition of its kinase activity.
David D. Ginty, Harvard Medical School
Ret (ret proto-oncogene) is a common receptor for the glial-derived neurotrophic factor (GDNF) family of ligands that is highly expressed in nonpeptidergic neurons as well as many other cell types.
Exons 14 and 15 are flanked by loxP sites in this Retfl targeted mutant strain. A V805A mutation in exon 15 is functionally silent, but sensitizes the protein to chemical inhibition of its kinase activity; signaling should be blocked by 1NMPP1 (unpublished). Cre-mediated excision of the floxed region results in a knockout allele.
When crossed with Wnt1-Cre mice, RET expression is undetectable in dorsal root ganglia (DRG), indicating efficient cre-mediated excision of the floxed region. Within 3 days of birth the compound mutant mice show abdominal distension, progressive weakness, and few survive beyond 3 weeks of age. Examination at postnatal day 14 (P14) reveals that they have an enlarged jejunum, ileum, and colon, suggesting that they may have intestinal aganglionosis similar to human patients carrying RET mutations who develop Hirschsprung's disease.
This allele places exon 14, exon 15 (modified to incorporate a V805A mutation involving a central T to C nucleotide change in the amino acid), and an FRT-flanked neomycin resistance cassette between loxP sites. The allele was created via homologous recombination in 129.1 embryonic stem (ES) cells. Resultant chimeric mice were crossed with B6/SJL background Tg(ACTFLPe)9205Dym animals (see Stock No. 003800) to excise the neomycin cassette. This strain was backcrossed to C57BL/6J for at least 10-15 generations by the donating lab.
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Two of the 27 markers throughout the genome were segregating, and 1 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were not completely backcrossed.
|Allele Name||targeted mutation 1.2, David D Ginty|
|Allele Type||Targeted (Conditional ready (e.g. floxed), No functional change)|
|Gene Symbol and Name||Ret, ret proto-oncogene|
|Strain of Origin||129|
|Molecular Note||Exons 14 and 15 and an FRT-flanked neo cassette are flanked by loxP sites. A missense mutation in exon 15 changes valine 805 to alanine. This mutation is silent but sensitizes the protein to chemical inhibition of its kinase activity. Flp-mediated recombination removed the FRT-flanked neo cassette leaving exons 14 and 15 floxed.|
Heterozygous and homozygous floxed mice are viable and fertile.
When using the Retf mouse strain in a publication, please cite the originating article(s) and include JAX stock #028548 in your Materials and Methods section.