B6NCrl.129P2(B6)-Apoe^tm1Unc Ccr2^tm1.1Blck Ccr5^tm3.1Blck/BlckJ

Stock number: 030896
Product name: 25AE-Bd
Research areas: Cardiovascular Research, Mouse/Human Gene Homologs, Research Tools

Description

25AE-Bd mice are C57BL/6NCrl-congenic triple mutant animals harboring the ApoE knock-out allele (Apoe^tm1Unc), the CCR2 knock-out allele (Ccr2^tm1.1Blck) and the CCR5 knock-out allele (Ccr5^tm2.1Blck). These mice are useful for studying the pathogenesis and treatment of atherosclerosis, as well as lipoprotein/cholesterol metabolism, neurovascular function, inflammation and neurodegenerative cardiovascular disease.

Detailed description

C57BL/6N-congenic 25AE-Bd mice homozygous for knock-out alleles of ApoE, Ccr2 and Ccr5 (ApoE-/-Ccr2-/-Ccr5-/-) are viable and fertile with no apparent reproductive abnormalities.

Of note, the Ccr2 knock-out allele (Ccr2^tm1.1Blck) has an EGFP replacing most of the Ccr2 coding region. The donating investigator reports that mice homozygous for this Ccr2 knock-out allele exhibit EGFP fluorescence in blood monocytes (which are CCR2+) via flow cytometry and and fluorescence microscopy.

In addition, the Ccr5 knock-out allele (Ccr5^tm2.1Blck) has a DsRed2 replacing the Ccr5 coding region. The donating investigator reports that mice homozygous for this Ccr5 knock-out allele exhibit DsRed2 fluorescence in bone marrow-derived macrophages via fluorescence microscopy, and no DsRed2 activity was observed in blood via flow cytometry. [Luckow et al. 2009 Genesis 47:545]

Dr. Bruno Luckow has made the following C57BL/6N-congenic strains available: ApoE knock-out (AE-B; Stock No. 030891), ApoE Ccr1 double knock-out (1AE-B; Stock No. 030892), ApoE Ccr2 double knock-out (2AE-Bd; Stock No. 030893), ApoE Ccr5 double knock-out (5AE-B; Stock No. 030894), ApoE Ccr1 Ccr5 triple knock-out (15AE-B; Stock No. 030895) and ApoE Ccr2 Ccr5 triple knock-out (25AE-Bd; Stock No. 030896).

Development

These 25AE-Bd mice (Stock No. 030896) are C57BL/6NCrl-congenic triple mutant animals harboring the ApoE knock-out allele (Apoe^tm1Unc), the CCR2 knock-out allele (Ccr2^tm1.1Blck) and the CCR5 knock-out allele (Ccr5^tm2.1Blck). Each mutant allele is described below.

The ApoE knock-out allele (Apoe^tm1Unc) was created in the laboratory of Dr. Nobuyo Maeda (University of North Carolina at Chapel Hill) to have a neo cassette replacing part of the coding region of the apolipoprotein E Locus on chromosome 7. In 2005, Dr. Bruno Luckow (Klinikum der Universitaet Muenchen) obtained C57BL/6J-congenic ApoE knock-out mice from The Jackson Laboratory (Stock No. 002052), bred them several generations to C57BL/6NCrl mice (Charles River Laboratories), and then used them as described below.

The CCR2 knock-out allele (Ccr2^tm1.1Blck) was created in the laboratory of Dr. Bruno Luckow (Klinikum der Universitaet Muenchen) to have an enhanced green fluorescent protein gene (EGFP) replacing most of the coding region of the chemokine (C-C motif) receptor 2 locus on chromosome 9. First, the original targeting vector EGFP::loxP-neo-loxP::polyA was electroporated into 129P2/OlaHsd-derived E14.1 embryonic stem (ES) cells. Correctly targeted ES cell clone L112 was used as described below.
The CCR5 knock-out allele (Ccr5^tm2.1Blck) was created in the laboratory of Dr. Luckow to have a red fluorescent protein gene (DsRed2) replacing the coding region of the chemokine (C-C motif) receptor 5 locus on chromosome 9. First, the original targeting vector DsRed2::loxP-PGK-hygro-loxP::polyA was electroporated into E14.1 ES cell clone L112 already targeted to have EGFP::loxP-neo-loxP::polyA inserted into the Ccr2 locus (see above). Chimeras were bred to C57BL/6J mice for germline transmission. The floxed selection cassettes in each knock-out allele have been permanently removed by exposure to Cre recombinase - resulting in the CCR2/CCR5 double mutant colony (Ccr2^tm1.1Blck Ccr5^tm2.1Blck). The CCR2/CCR5 double mutant colony was backcrossed ten generations to C57BL/6NCrl, and then used as described below.
Of note, the Ccr2 and Ccr5 loci are ~8 kbp apart on chromosome 9 [GRCm38.p5].

To generate the ApoE/CCR2/CCR5 triple mutant colony [25AE-Bd], Dr. Luckow bred these mouse lines together. The donating investigator reports that the 25AE-Bd colony was backcrossed to C57BL/6NCrl for a total of at least five generations. In 2017, Dr. Luckow sent 25AE-Bd mice to The Jackson Laboratory as Stock No. 030896. Upon arrival, sperm was cryopreserved.
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6NCrl inbred male (thus the Y chromosome of the congenic strain is of C57BL/6NCrl origin).

In 2017, a SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6N and C57BL/6J substrains, was performed on the male mice sent to The Jackson Laboratory Repository. All markers were C57BL/6 allele-type. In addition, all markers that determine C57BL/6 substrains were C57BL/6N allele-type with one exception - the marker on chromosome 6 [~107.8 Mbp] was found to be homozygous for C57BL/6J allele-type. This suggests that C57BL/6NCrl allele-type at this marker is the same as C57BL/6J allele-type (rather than C57BL/6NJ allele-type).

Allele

Symbol: Ccr2^tm1.1Blck
Name: targeted mutation 1.1, Bruno Luckow
MGI accession ID: MGI:6272721
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Ccr2
Marker name: chemokine (C-C motif) receptor 2
Chromosome: 9
Strain of origin: 129P2/OlaHsd
Molecular note: Most of the open reading frame is replaced by an EGFP reporter gene and a floxed neomycin resistance cassette. Cre-mediated recombination removed the floxed neo cassette.
General note: The version of this allele that contains the neomycin selection cassette, Ccr2^tm1Blck, has been analyzed.

Allele

Symbol: Apoe^tm1Unc
Name: targeted mutation 1, University of North Carolina
MGI accession ID: MGI:1857129
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Apoe
Marker name: apolipoprotein E
Chromosome: 7
Strain of origin: 129P2/OlaHsd
Molecular note: Insertion of a neomycin resistance cassette deleted part of exon 3 and part of intron 3 of the Apoe gene. Plasma from homozygous mutant mice gave no detectable immunoprecipitate by the Ouchterlony double immunodiffusion test using a rabbit antibody to rat APOE.

Allele

Symbol: Ccr5^tm3.1Blck
Name: targeted mutation 3.1, Bruno Luckow
MGI accession ID: MGI:6272797
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Ccr5
Marker name: chemokine (C-C motif) receptor 5
Chromosome: 9
Strain of origin: 129P2/OlaHsd
Molecular note: The coding region was replaced with DsRed2 and a floxed hygro cassette. Cre-mediated recombination removed the floxed hygromycin cassette.
General note: The allele was made in ES cell line that carries Ccr2^tm1.1Blck.