The Raf1loxP/loxP targeting vector contains a conditional mutant form of Raf1. After cre-mediated recombination these mice may be useful for studying facial abnormalities and hypertrophic cardiomyopathy associated with the autosomal dominant disorder Noonan syndrome (NS).
Benjamin Neel, NYU School of Medicine
Mice homozygous for the Raf1loxP/loxP allele are viable and fertile. This allele contains a floxed sequence containing a cDNA fragment encoding v-raf-leukemia viral oncogene 1 (Raf1) wild-type exons 13 to 16 and a neomycin (neo) resistance cassette downstream of exon 12 of the Raf1 gene. A point mutation was introduced downstream of the floxed sequence, resulting in the missense mutation D486N, associated with the autosomal dominant disorder Noonan syndrome (NS). Raf1 is a MAP kinase kinase kinase (MAP3K), which is activated by Ras GTPases, which in turn activates MEK1/2 and extracellular signal-regulated kinase (ERK)1/2 pathways. Activated ERKs are involved in the cell division cycle, apoptosis, cell differentiation and cell migration. The D486N, kinase-impaired, mutation is associated with the autosomal dominant disorder Noonan syndrome (NS) characterized by short stature, facial dysmorphia, cardiovascular abnormalities, and myeloproliferative disease (MPD). Homozygous mice are viable and fertile. When these mutant mice are bred to mice that express Cre recombinase, resulting offspring will have the floxed sequence deleted in cre-expressing tissues.
For example, when bred to mice expressing germ line cre (see B6.FVB-Tg(EIIa-cre)C5379Lmgd/J Stock No. 003724), removal of the floxed sequence permits expression of mutant Raf1D486N. Heterozygous D486N/+ females exhibit a mild growth defect. One-third of homozygous mice had body length and weight only 50% that of littermate controls. A majority of these smaller mice (s-D486N/D486N) die shortly after weaning, while survivors only live 4 and 8 months. The longer-lived s-D486N/D486N mice exhibit reduced body size, a hunched appearance with ruffled fur, and frequent tremors. These mice have a slight decrease in skull width resulting in a ?triangular? facial appearance. The mice with normal length and weight (n-D486N/D486N) display an increase in heart weight/body weight ratio with an increase also seen in the left ventricular diastolic posterior wall thickness. They exhibit a decrease in left ventricular internal endsystolic dimension, and an increase in stroke volume, fractional shortening, cardiac output, and ejection fraction. Raf1D486N-expressing cells show enhanced ERK/MAPK pathway activation and increased heterodimerization with BRAF.
A targeting construct was designed to insert a loxP site, a splice acceptor sequence, a cDNA fragment encoding v-raf-leukemia viral oncogene 1 (Raf1) wild-type exons 13 to 16, and a neomycin (neo) resistance cassette followed by a second loxP site downstream of exon 12 of the Raf1 gene. A point mutation was introduced in exon 13 resulting in the missense mutation D486N, associated with the autosomal dominant disorder Noonan syndrome (NS). This targeting construct was electroporated into (129S6/SvEvTac x C57BL/6NCr)F1-derived G4 embryonic stem (ES) cells and correctly targeted ES cells were aggregated with (129Sv x C57BL/6)F1 morulae. Resulting chimeric blastocysts were implanted in pseudopregnant females. Male offspring were crossed to C57BL/6 females. These mice were maintained on a mixed background. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.
|Allele Name||targeted mutation 2, Toshiyuki Araki|
|Gene Symbol and Name||Raf1, v-raf-leukemia viral oncogene 1|
|Strain of Origin||(129S6/SvEvTac x C57BL/6NCrl)F1|
|Molecular Note||A targeting vector containing a loxP site, cDNA spanning exons 13 through 16, neo cassette, second loxP site and modified exon 13 was inserted into intron 12. The modified exon 13 contains nucleotide substitution(s) that result in the amino acid substitution of asparagine for aspartic acid at position 486 (D486N).|
When maintaining a live colony, homozygous mice may be bred together.
When using the B6;129-Raf1tm2Ara/J mouse strain in a publication, please cite the originating article(s) and include JAX stock #021233 in your Materials and Methods section.