B6;129S-Igk^tm5.1Fwa Igh^tm16.1Fwa/J

Stock number: 041904
Product name: PCT64
Strain synonyms: PCT64
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools, Virology Research

Description

PCT64 mice carry humanized modifications of the mouse heavy (Igh) and light (Igk) immunoglobulin chain regions to enable the testing of broadly neutralizing antibody (bnAb) responses to the conserved PCT64 V2 apex HIV-1 envelope protein immunogen.

Detailed description

Broadly neutralizing antibodies (bnAbs) recognize conserved targets on different human immunodeficiency virus-1 (HIV-1) strains and can inhibit a wide spectrum of viral variants. To induce bnAbs, vaccines must be highly selective for rare human antibodies that can develop into bnAbs.

This PCT64 strain is part of a panel of humanized mouse models that cover the major sites of vulnerability on the HIV-1 envelope protein, including V3 glycan (DH270, Stock No. 041901 and BG18, Stock No. 041902), V2 apex (PG9, Stock No. 041903 and PCT64, Stock No. 041904), Membrane Proximal External Region (MPER) (DH511, Stock No. 041905), and CD4 binding site (CH235, Stock No. 041906).

These models can produce diverse potential precursors for HIV-1 bnAbs in complex antibody repertoires which mimic human vaccination conditions. As such, the mice can be used to assess and optimize vaccine candidates at the pre-clinical stage.

Development

The mouse light (Igk) and heavy (Igh) immunoglobulin chain regions were modified to incorporate human sequences to allow the testing of broadly neutralizing antibody (bnAb) responses to conserved HIV-1 envelope protein immunogens. The Igk and Igh mutations were independently generated through homologous recombination in 129S/SvEv x C57BL/6-derived EF1 embryonic stem (ES) cells. Strains bearing the mutations were intercrossed to create compound mutant animals. The donating laboratory maintained the mice on a mixed 129S and C57BL/6 genetic background. LoxP-flanked neomycin cassettes in each of the targeting constructs were removed from ES cell clones through the transient expression of Adeno-cre recombinase. Upon arrival, sperm was cryopreserved. To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

To express bnAb precursor light chains (LCs), modifications were made to the mouse Igk region on Chromosome 6: the germline human V segment (hV_κ; IGKV3-20*01) of a bnAb LC replaces the mouse IGKV3-7*01 segment and is associated with a 12-base pair spacer (12-RSS), as are the mV_k segments. The intact mouse J_κ (mJ_k) region retains its endogenous 23-RSS. 

Although a CRISPR/cas9-generated CAG-hTdT-IRES-EGFP cassette expressing human terminal deoxynucleotidyl transferase (hTdT; formal gene symbol DNTT, DNA nucleotidylexotransferase) cDNA was originally introduced to intron 1 of the mouse Gt(ROSA)26Sor gene downstream of the mutant Igk allele on Chromosome 6, this feature was lacking in the final mouse model. The loss of this allele potentially occurred due to a meiotic crossover event separating the Igk and Gt(ROSA)26Sor alleles.

To express bnAb precursor heavy chains (HCs), modifications were made to the mouse Igh region on Chromosome 12: the mouse V_H5-2 (Ighv5-2) segment was replaced with human V_H (IGHV3-15*01); the mouse intergenic control region 1 (IGCR1, located between the V_H and D clusters) was deleted; and all four of the mouse J_H (mJ_H) segments were replaced with a single human J_H (IGHJ6*03). A human D segment (IGHD3-3*01) was placed immediately upstream of the hJ_H, in place of mouse DQ52.

Allele

Symbol: Igk^tm5.1Fwa
Name: targeted mutation 5.1, Frederick W Alt
MGI accession ID: MGI:8394056
Generation method: Targeted
Attributes: Humanized sequence
Marker symbol: Igk
Marker name: immunoglobulin kappa chain complex
Marker synonyms: kappa
Chromosome: 6
Strain of origin: 129S/SvEv x C57BL/6
Molecular note: The mouse light (Igk) immunoglobulin chain regions were modified to incorporate human sequences to enhance the testing of broadly neutralizing antibody (bnAb) responses to conserved HIV-1 envelope protein immunogens. The mouse Igk region on Chromosome 6: the germline human V segment (hVκ; IGKV3-20*01) of a bnAb LC was incorporated 3' of the mouse Vκ (mVk) cluster and is associated with a 12-base pair spacer (12-RSS), as are the mVk segments. The intact mouse Jκ (mJk) region retains its endogenous 23-RSS. LoxP-flanked neomycin cassettes in each of the targeting constructs were removed from ES cell clones through the transient expression of Adeno-cre recombinase.

Allele

Symbol: Igh^tm16.1Fwa
Name: targeted mutation 16.1, Frederick W Alt
MGI accession ID: MGI:8394057
Generation method: Targeted
Attributes: Humanized sequence
Marker symbol: Igh
Marker name: immunoglobulin heavy chain complex
Chromosome: 12
Strain of origin: 129S/SvEv x C57BL/6
Molecular note: The mouse heavy (Igh) immunoglobulin chain regions were modified to incorporate human sequences to enhance the testing of broadly neutralizing antibody (bnAb) responses to conserved HIV-1 envelope protein immunogens. The mouse Igh region on Chromosome 12: the mouse VH5-2 (Ighv5-2) segment was replaced with human VH (IGHV3-15*01); the mouse intergenic control region 1 (IGCR1, located between the VH and D clusters) was deleted; and all four of the mouse JH (mJH) segments were replaced with a single human JH (IGHJ6*03). A human D segment (IGHD3-3*01) was placed immediately upstream of the hJH, in place of mouse DQ52. LoxP-flanked neomycin cassettes in each of the targeting constructs were removed from ES cell clones through the transient expression of Adeno-cre recombinase.