B6(Cg)-Igk^{tm3.1(VRCO1)Nemz} Igh^{tm3.1(VRC01)Nemz}/J

Stock number: 035705
Product name: VRC01 HHLL; VRC01^gHL

Strain synonyms: VRC01^gHL
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools, Virology Research

Description

VRC01^gHL knock-in mice express both the germline-reverted (unmutated ancestor) light and heavy chains of the broadly neutralizing HIV antibody VRC01, which recognizes the CD4 binding site of the HIV viral envelope glycoprotein (Env).These mice are useful in studying the 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^gHL knock-in alleles have both the endogenous mouse immunoglobulin heavy and light (kappa) chain complex genes (Igh and Igk, respectively) replaced with a germline-reverted (unmutated ancestor) heavy and light chains of the broadly neutralizing HIV antibody VRC01, which recognizes a region of the HIV viral envelope glycoprotein (Env). The HIV VRC01 antibody H chain variable exons replace the respective J clusters in the endogenous mouse Igh gene. Similarly, the HIV VRC01 L chain variable exons including the mature LCDR3 (light chain complementarity determining region 3) replace the J clusters in the endogenous mouse Igk gene. The donating investigator reports double homozygous mice are viable and fertile with no apparent spontaneous defects.  VRC01^gHL mice exhibit normal B cell development and express surface B cell receptors similar to wild type mice. Approximately 33% of B cells from heterozygous mice and 90% of B cells from homozygous mice bind VRC01-class eOD-GT8 (a 60 mer immunogen designed to bind to inferred germline versions of VRC01-class B cell receptors).  Endogenous mouse light chains are also expressed on a high fraction of B cells. VRC01^gHL knock-in mice may be used to test VRC01-class B cell responses at physiologically relevant B cell precursor frequencies.

Development

The VRC01 gL and gH knock-in alleles (Igk^{tm3.1(VRCO1)Nemz} Igh^{tm3.1(VRC01)Nemz}, respectively) were created in the laboratory of Dr. David Nemazee (The Scripps Research Institute).

The VRC01^gH 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. 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. 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 B6.FVB-Tg(EIIa-cre)C5379Lmgd/J (Stock No. 003724) for germline deletion of the neo cassette. The resulting VRC01 gH mice were bred with C57BL/6J mice for at least 5 generations (and the cre-expressing transgene was removed) and then made homozygous.

The VCR01^gL targeting vector was constructed using a loxP-flanked neomycin cassette and the HIV antibody germline-reverted VRC01 sequence including the mature LCDR3 (light chain complementarity determining region 3) to replace the J cluster from Igk on chromosome 6. The targeting construct was electroporated into C57BL/6N-derived C2 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 B6.FVB-Tg(EIIa-cre)C5379Lmgd/J (Stock No. 003724) for germline deletion of the neo cassette. The resulting VRC01 gH mice were bred with C57BL/6J mice for 10 generations (and the cre-expressing transgene was removed) and then made homozygous. Homozygous VCR01^gH and VCR01^gL were bred together to create VCR01^gHL mice. Upon arrival, mice were bred to C57BL/6J for at least 1 generation to establish the colony.

Allele

Symbol: Igk^{tm3.1(VRCO1)Nemz}
Name: targeted mutation 3.1, David Nemazee
MGI accession ID: MGI:6459233
Generation method: Targeted
Attributes: Inserted expressed sequence
Synonyms: VRC01^gL
Marker symbol: Igk
Marker name: immunoglobulin kappa chain complex
Marker synonyms: kappa
Chromosome: 6
Strain of origin: C57BL/6NTac
Molecular note: The targeting vector was constructed using a loxP-flanked neomycin cassette and the HIV antibody germline-reverted VRC01 sequence including the mature LCDR3 (light chain complementarity determining region 3) to replace the J cluster from Igk. Cre-mediated recombination removed the floxed neo cassette.

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.