This mouse colony harbors three mutations: a p53- mutation, the Brca1F22-24 mutation, and a BLG-Cre transgene. The description of each is below.
To generate the p53 mutant allele, a targeting vector was designed to replace part of exon 5 of the mouse p53 tumor-suppressor gene, p53, with a PolII-neomycin (neo) resistance cassette (Harvey et al, Nat Gen, 1993; 5; 225-29). This construct was electroporated into 129S7/SvEvBrd-Hprt1+-derived AB1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and the resulting chimeric males were bred to C57BL/6 females. These mice were intercrossed to establish a colony of p53 transgenic mice.
To generate the Brca1F22-24 mutant allele, a targeting vector was designed by Dr. Alan Ashworth (Breakthrough Breast Cancer Research Centre, London) to replace exons 22-24 of the mouse breast cancer 1 gene (Brca1) with a loxP site, a cDNA sequence encoded by exons 22-24, a 3' myc epitope, bovine growth hormone polyA signal, a second loxP site, a splice acceptor sequence, and a PGK-neo cassette. This construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl+-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts and the resulting chimeric mice were bred to C57BL/6 mice. These mice were intercrossed to establish a colony of Brca1F22-24/F22-24 mice on a mixed background.
The BLG-Cre transgene was designed by Dr. Alan R. Clarke (while at University Medical School, Edinburgh, United Kingdom) to have Cre recombinase under the control of the sheep mammary gland specific promoter of the ovine beta-lactoglobulin (BLG) gene. The construct included exon 1 of sheep BLG, and a Cre recombinase with a modified initiation codon and a polyadenylation signal (polyA). The construct was microinjected into the pronuclei of (CBA x C57BL/6) fertilized oocytes, and mice from founder line 74 were identified and bred to C57BL/6 mice. These mice were intercrossed to establish a colony of BLG-cre mice on a mixed background.
Mutant mice were bred together to generate the triple mutant colony. Mice heterozygous for the p53 allele, homozygous for the floxed Brca1 allele (Brca1F22-24/F22-24), and hemizygous for the BLG-cre transgene (also called BLG-Cre; Brca1F22-24/F22-24; p53+/- mice) were maintained on a mixed genetic background prior to sending to The Jackson Laboratory Repository. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.
