B6(Cg)-Igh^{tm3.1(VRC01)Nemz}/J

Stock number: 029584
Product name: VRC01 gH knock-in
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools, Virology Research

Description

VRC01 gH knock-in mice express a germline-reverted (unmutated ancestor) heavy chain of the broadly neutralizing HIV antibody VRC01, which recognizes a region of the HIV viral envelope glycoprotein (Env) and retains the ability to pair with native mouse IgL chains. These mice are useful in studying the protective role of vaccine-induced broadly neutralizing antibodies (bnAbs) against primary HIV infection.

Detailed description

A minority of HIV-infected individuals eventually develop antibodies capable of neutralizing the majority of the diverse HIV strains circulating in the human population. Such broadly neutralizing antibodies (bnAbs) generally manifest only after years of infection and do not control virus in the individuals in which they arose - but recombinant forms of such bnAbs can prevent infection in animal models. Vaccine-induced generation of bnAbs may be protective against primary HIV infection. The CD4 binding site (CD4bs) antibody VRC01 and other VRC01-class bnAbs have been identified in at least seven different HIV-positive donors, and might be amenable to reproducible vaccine elicitation.

The VRC01 gH knock-in allele has the endogenous mouse immunoglobulin heavy chain complex gene (Igh) replaced with a germline-reverted (unmutated ancestor) heavy chain of the broadly neutralizing HIV antibody VRC01, which recognizes a region of the HIV viral envelope glycoprotein (Env) and retains the ability to pair with native mouse IgL chains. The HIV VRC01 antibody H chain variable exons replace the respective J clusters in the endogenous mouse Igh gene. The donating investigator reports homozygous mice are viable and fertile with no apparent spontaneous defects.
Induced antibodies showed characteristics of VRC01-class bnAbs, including a short CDRL3 (light-chain complementarity-determining region 3) and mutations that favored binding to near-native HIV-1 gp120 constructs.

Development

The VRC01 gH knock-in allele (Igh^{tm3.1(VRC01)Nemz}) was created in the laboratory of Dr. David Nemazee (The Scripps Research Institute). The targeting vector was designed to replace the DQ52-JH cluster in the immunoglobulin heavy chain complex gene (Igh) on chromosome 12 with a loxP-flanked neomycin resistance cassette and the HIV broadly neutralizing antibody (bnAb) precursor VRC01 gH VDJ exon (more details below). The upstream promoter, leader and intron elements were from VHJ558.85.191. The targeting construct was electroporated into C57BL/6-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric mice were bred with albino C57BL/6 to establish the colony. Offspring were bred with EIIa-Cre mice (on a C57BL/6 background; see Stock No. 003724) for germline deletion of the neo cassette. The resulting VRC01 gH mice were bred with C57BL/6J mice for several generations (and the cre-expressing transgene was removed), and then maintained as homozygotes prior to sending males with black coat color to The Jackson Laboratory Repository in 2016. 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).
Of note, it is not known if the Y chromosome has been fixed to the C57BL/6 background during backcrossing.
Although the true germline precursor is not known for VRC01 (or other VRC01-class bnAbs), Dr. Nemazee's group approximated the true immunoglobulin heavy (H) chain precursor with a VRC01 germline-reverted H chain (VRC01 gH; composed of the VH1-2*02 and IGHJ1*01 genes), along with the complementarity-determining region 3 of the H chain (CDRH3) from VRC01 with a single mutation to remove an unpaired cysteine.

Allele

Symbol: Igh^{tm3.1(VRC01)Nemz}
Name: targeted mutation 3.1, David Nemazee
MGI accession ID: MGI:5779356
Generation method: Targeted
Attributes: Inserted expressed sequence
Synonyms: VRC01 gH
Marker symbol: Igh
Marker name: immunoglobulin heavy chain complex
Chromosome: 12
Strain of origin: C57BL/6
Molecular note: A floxed neomycin resistance cassette replaced the DQ52-JH cluster in the Igh locus with the HIV broadly neutralizing antibody precursor VRC01 gH VDJ exon. The upstream promoter, leader and intron elements were from VHJ558.85.191. Cre-mediated recombination removed the neomycin resistance cassette.