B6J.129(Cg)-Brca1^tm3.1Rjbr/Mmjax

Stock number: 036739
Product name: Brca1^FH-I26A
Research areas: Cancer Research, Cell Biology Research, Metabolism Research

Description

Brca1^FH-I26A knock-in mice carry an I26A (isoleucine to alanine) amino acid substitution in the Brca1 (breast cancer 1, early onset) gene that abrogates E3 ligase activity. These mice may be useful for studying genome maintenance and breast cancer.

Detailed description

Brca1 (breast cancer 1, early onset) encodes a nuclear phosphoprotein that functions as a tumor suppressor and is involved in maintaining genomic stability. BRCA1 polypeptides exist as a heterodimeric complex with BARD1, the subsequent heterodimer functions as a ubiquitin E3 ligase.

Brca1^FH-I26A knock-in mice carry FLAG and HA epitope tags (FH) and a I26A (isoleucine to alanine) amino acid substitution in the RING domain. The mutation abolishes the ubiquitin E3 ligase activity of BRCA1 by disrupting its interaction with ubiquitin E2 conjugases. The mutation does not disturb the overall structure of the RING domain or prevent the formation of the BRCA1/BARD1 heterodimer. Homozygous females are viable and fertile, males are viable, but are infertile. Homozygous Brca1^FH-I26A mice exhibit a decreased (5-10%) body weight and in males, spermatogenesis is arrested - seminiferous tubules lack elongated spermatids and spermatozoa. Homozygous mice do not exhibit increased tumor susceptibility.

Of note, the control strain, Brca1^FH-WT is available as B6;129-Brca1^tm4.1Rjbr/Mmjax (MMRRC Stock No. 069514)

Development

The Brca1^FH-I26A knock-in allele was created by Dr. Richard Baer at Columbia University. The targeting vector was designed to insert a fragment containing modified exons 1 and 2 with an FRT-flanked promoterless hygromycin resistance sequence in intron 1. Sequence encoding an artificial methionine and the FLAG and HA epitope tags (FH; (MDYKDDDDKYPYDVPDYA) was inserted upstream of the natural translation initiation codon. In addition, an isoleucine to alanine substitution was introduced at position 26 (I26A) in the RING domain of exon 2 of the Brca1 gene on chromosome 11. Thus, the allele encodes a full-length Brca1 polypeptide (1812 amino acids) with an I26A mutation fused to an N-terminal FH epitope. The construct was electroporated into 129Sv-derived unspecified embryonic stem (ES) cells. Transient transfection with a Pgk-Flpe plasmid removed the hygromycin cassette. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were bred to C57BL/6 females and the mutation was confirmed by sequence analysis. Mice were backcrossed to C57BL/6J mice for 4 generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Brca1^tm3.1Rjbr
Name: targeted mutation 3.1, Richard J Baer
MGI accession ID: MGI:5494435
Generation method: Targeted
Attributes: Not Applicable
Synonyms: Brca1^FH-I26A
Marker symbol: Brca1
Marker name: breast cancer 1, early onset
Marker synonyms: BRCA-1; BRCAI; BRCC1; BROVCA1; FANCS; IRIS; PNCA4; PPP1R53; PSCP; RNF53
Chromosome: 11
Strain of origin: 129
Molecular note: Exon 2 was replaced with a modified exon 2 containing an artificial initiator methionine, FLAG and HA epitope tags and nucleotide substitutions that result in the amino acid substitution of alanine for isoleucine at position 26 (I26A). This mutation results in an E3 ligase enzymatically defective protein. Flp-mediated recombination removed a FRT-flanked hygromycin resistance cassette inserted upstream of the modified exon 2.