B6.129P2(Cg)-Braf^tm1Mmcm/J

Stock number: 017837
Product name: BRaf^CA
Strain synonyms: BRAF^F-V600E
Research areas: Cancer Research, Cell Biology Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

BRaf^CA is a knock-in allele of human BRaf which expresses normal BRaf prior to Cre recombinase exposure. Cre-mediated recombination results in the BRaf^VE allele, which expresses the constitutively active BRAF^V600E mutant protein. In conjunction with Cre-expressing mouse lines, these BRaf^CA mice may be useful in studies of human malignancies, as well as for studying the RAS-activated RAF/MEK/ERK mitogen-activated protein kinase signaling pathway and pleiotropic regulators of the aberrant cancer cell physiology.

Detailed description

Mice homozygous for the Cre-activated BRaf mutant allele (BRaf^CA) are viable and fertile. Mice express the transcript encoded by endogenous exons 1-14 and loxP-flanked human exons 15-18 (BRaf^CA) prior to Cre recombinase exposure. Homozygous mice have "normal" BRaf expression, and no abnormalities are reported for homozygous BRaf^CA mice.

Following Cre-mediated excision of the floxed sequences (human BRAF exons 15-18 and polyA signal), the transcripts are subsequently generated using the downstream mutant exon 15 (modified with a V600E amino acid substitution associated with constitutively active BRAF^V600E in human cancers) and the endogenous downstream exons 16-18. The resulting BRaf^VE transcripts are subject to normal patterns of alternate splicing and differential exon usage, and BRaf^VE expression is observed at physiological levels. To discriminate the expression of the various BRaf mRNAs in heterozygous mice, silent restriction enzyme polymorphisms were incorporated into the exon 15 sequences of BRaf^CA (BamHI) and BRaf^VE (XbaI).

For example, when crossed to a strain expressing tamoxifen-inducible Cre recombinase (see Stock No. 017585), this mutant mouse strain may be useful in studies of lung tumors.

Development

The BRaf^CA mutant mice were created by Dr. Martin McMahon (University of California, San Francisco). The BRaf^CA targeted mutation was designed to replace the endogenous Braf exon 15 with (from 5' to 3') a loxP site, the cDNA sequence encoded by human BRAF exons 15-18 (with a silent BamHI restriction site polymorphism in exon 15), the mouse Braf polyA sequences, a PGK-neo cassette (with triple transcriptional stop and SV40 polyA signal), a second loxP site, and a mouse exon 15 sequence modified via site-directed mutagenesis to harbor both a V600E amino acid substitution (associated with the constitutively active BRAF^V600E mutation in human cancers) and also a silent XbaI restriction site polymorphism. The targeting vector was microinjected into 129Sv/OLA embryonic stem (ES) cells, and correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric mice were bred with FVB/N mice to establish the colony. BRaf^CA mutant mice were subsequently maintained on a C57BL/6 congenic background.
This BRaf^CA strain was established at The Jackson Laboratory in 2012 from the same project that assembled the BRaf^CA Pten^loxP Tyr::CreER^T2 triple mutant line (Stock No. 013590). Mice identified by genotype with the BRaf^CA allele, without the Pten^loxP allele and without the Tyr::CreER^T2 transgene (confirmed September 2015) were used as founders for this BRaf^CA strain (Stock No. 017837).

Allele

Symbol: Braf^tm1Mmcm
Name: targeted mutation 1, Martin McMahon
MGI accession ID: MGI:3711771
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Braf
Marker name: Braf transforming gene
Chromosome: 6
Strain of origin: 129P2/OlaHsd
Expressed genes: BRAF,B-Raf proto-oncogene, serine/threonine kinase,human
Site of expression: intranasal instillation of an adenovirus expressing Cre recombinase results in benign lung tumors that only rarely progress to adenocarcinoma.
Molecular note: A targeting vector was designed to insert a loxP site and the human BRAF cDNA covering exons 15-18 into intron 14, along with a floxed neo cassette. A modified exon 15 was engineered into the vector to encode Braf^V600E and a silent XbaI restriction site. RT-PCR confirmed that mutant transcript was expressed at the same level as wild-type, and that the V600E mutation was not detected.