B6.129P2(C)-Igh^tm2Cgn/J

Stock number: 012642
Research areas: Immunology, Inflammation and Autoimmunity Research

Description

These B1-8i mutant mice carry two copies of V_HD_HJ_H gene arrangements in their immunoglobulin heavy chain complex (IgH) alleles, representing a recombined antibody variable region derived from a 4-hydroxy-3-nitrophenylacetyl binding antibody (B1-8), replacing an endogenous IgH-D element (DQ52) and the IgH-J elements. This strain may be useful for studying the effect of a restricted antibody repertoire on immune responses.

Detailed description

These B1-8i mutant mice carry two copies of V_HD_HJ_H gene arrangements in their immunoglobulin heavy chain complex (IgH) alleles, representing a recombined antibody variable region derived from a 4-hydroxy-3-nitrophenylacetyl binding antibody (B1-8), replacing an endogenous IgH-D element (DQ52) and the IgH-J elements. A silent point mutation in codon 92 inactivates the internal heptameric recombination signal sequence (RSS), abolishing endogenous V_H replacement and increasing the stability of the gene segment. Mice homozygous for this allele are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. This strain may be useful for studying the effect of a restricted antibody repertoire on immune responses.

Development

The B1-8VDJ targeting vector was designed to replace endogenous sequence encompassing a 3' Igh-D element (DQ52) and the Igh-J elements of immunoglobulin heavy chain complex (IgH) with a recombined antibody variable region derived from a 4-hydroxy-3-nitrophenyl acetyl binding antibody (B1-8). The B1-8 variable region was modified to contain a point mutation in codon 92, inactivating an internal heptameric recombination signal sequence. A loxP-flanked neomycin resistance (neo) cassette was inserted upstream of the B1-8 region. The construct was electroporated into 129P2/OlaHsd-derived E14.1 embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a pIC-Cre expression plasmid to delete the floxed-neo cassette, and were injected into CB.20 (C57BL/6 congenic with BALB/c at the IgH locus) blastocysts. The donating investigator stated that the resulting chimeric mice were bred to CB.20 mice and offspring were backcrossed to CB.20 (see SNP note below) at least 10 generations to establish a colony of homozygous B1-8i mice. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 4 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Igh^tm2Cgn
Name: targeted mutation 2, University of Cologne
MGI accession ID: MGI:2388486
Generation method: Targeted
Marker symbol: Igh
Marker name: immunoglobulin heavy chain complex
Chromosome: 12
Strain of origin: 129P2/OlaHsd
Molecular note: Homologous recombination and subsequent in vitro cre-mediated excision resulted in the replacement of endogenous sequence encompassing a 3' Igh-D element (DQ52) and the Igh-J elements with a recombined antibody variable region derived from a 4-hydroxy-3-nitrophenyl acetyl binding antibody (B1-8). The B1-8 variable region was modified to contain a point mutation in codon 92, inactivating the heptameric recombination signal sequence. Flow cytometry verified cell surface expression of the mutant protein product.
General note: The targeted IgH allele was of the a allotype.