129S(B6)-Igh^tm10.1Fwa/J

Stock number: 040898
Product name: IGCR1/CBE2^-
Research areas: Immunology, Inflammation and Autoimmunity Research

Description

IGCR1/CBE2^- mice are useful in studies of CTCF zinc finger protein-binding elements in the Igh (immunoglobulin heavy chain complex) locus. These animals carry a scrambled mutation of the CTCF binding site 2 (CBE2) site within the mouse Igh intergenic control region 1 (IGCR1).

Detailed description

Immunoglobulin heavy chain (IgH) variable region exons are assembled from variable (V), diversity (D), and joining (J) gene segments in developing B lymphocytes. The intergenic control region 1 (IGCR1) is a key Igh V(D)J recombination regulatory region which lies between the V_H and D clusters. The IGCR1 promotes normal B-cell development and balances antibody repertoires using CTCF protein-binding elements (CBEs). CTCF is an 11-zinc-finger nuclear protein implicated in transcriptional insulation, chromatin boundary formation, transcriptional activation/repression and chromosome looping.

IGCR1/CBE2^- mice carry a scrambled mutation in the 20 bp CTCF binding site 2 (CBE2) region of the mouse Igh IGCR1 that does not bind CTCF. The mutation is not expressed and is excised after V(D)J recombination.

Individual inactivation of CBE2 leads to partial impairment of various IGCR1 functions (e.g. B cell development, V_H transcription, Igh V(D)J rearrangements) relative to the far greater effects of inactivating both the CBE1 and CBE2 binding elements simultaneously (see Stock No. 040896). CBE1 and CBE2 function cooperatively to achieve full IGCR1 regulatory activity.

Mutation of CBE1 alone (see Stock No. 040897) appears, on average, to have a greater impact on repertoire, order, and feedback regulation in developing B cells than does mutation of CBE2 alone.

Development

A targeting vector was originally designed to introduce scrambled mutations of the divergently oriented 20-bp CTCF binding site 1 (CBE1) and 19-bp CBE2 sites within intergenic control region 1 (IGCR1) of the mouse Igh locus (NCBI accession number: AJ851868; see Stock No. 040896). Resultant animals carrying only the CBE2 mutation were chosen to create this IGCR1/CBE2^- strain (Stock No. 040898).

PCR site-directed mutagenesis was used to create the targeting vector carrying the scrambled CBE1 and CBE2 mutations: wildtype CBE1: 5'-GCTTCCCCCTTGTGGCCAT-3', mutant CBE1: 5'-CCTGCTAGCCTTCTCGTCG-3'; wildtype CBE2: 5'-TCTCCACAAGAGGGCAGAA-3', mutant CBE2: 5'-ACTAGTAAAAAGCGGCCGC-3'. These mutations incorporate restriction endonuclease recognition sites; NheI in CBE1 and SpeI in CBE2. A loxP-flanked neomycin cassette was introduced between the CBE1 and CBE2 regions. The targeting construct was electroporated into 129S6/SvEvTac-derived TC1 embryonic stem (ES) cells. Resultant clones carrying only the scrambled CBE2 sequence were subjected to adenovirus-mediated Cre recombinase deletion to remove the loxP-flanked neomycin gene. The donating investigator reported that the strain was initially backcrossed to both 129S6/SvEvTac and C57BL/6, then backcrossed to 129S6/SvEvTac for an unknown number of generations over a period of 5 years prior to sending agouti males to The Jackson Laboratory in 2025 (see SNP results below). Upon arrival, sperm was cryopreserved. To establish a live colony, an aliquot of frozen sperm was used to fertilize 129S1/SvImJ oocytes (Stock No. 002448).

In 2025, a SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome (previously shown to match for 129S6/SvEvTac isogenic mice and Stock No. 002448 129S1/SvImJ mice), as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory. This revealed a total of 2 markers that were not fixed for 129S1 allele-type (chromosomes 3 [~37.4 Mb], 12 [~30.0 Mb]). These data suggest that the mice sent to The Jackson Laboratory (and used to freeze sperm in 2025) may not have been completely backcrossed onto 129S.

Allele

Symbol: Igh^tm10.1Fwa
Name: targeted mutation 10.1, Frederick W Alt
MGI accession ID: MGI:5752253
Generation method: Targeted
Attributes: Not Applicable
Synonyms: IGCR1/CBE2^-
Marker symbol: Igh
Marker name: immunoglobulin heavy chain complex
Chromosome: 12
Strain of origin: 129S6/SvEvTac
Molecular note: In intergenic control region 1 (IGCR1) the second CCCTC-binding factor (CTCF)-binding element (CBE2) was replaced with scrambled sequence that does not bind CTCF. A floxed neo cassette was also inserted between the two CBEs and subsequently removed by cre mediated recombination.