B6;129S-Igk^tm4.1Fwa Gt(ROSA)26Sor^{tm4(CAG-DNTT)Fwa} Igh^tm15.1Fwa/J

Stock number: 041903
Product name: PG9
Strain synonyms: PG9
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools, Virology Research

Description

PG9 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 PG9 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 PG9 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), a recombination cassette was integrated into the mouse Igk region on Chromosome 6: germline human V (hV_λ; IGLV2-14*01) and J (hJ_λ; IGLJ3*01) replace all five mouse J_κ (mJ_k) segments. The hV_λ segment was associated with a recombination signal sequence containing a 23-base pair spacer (23-RSS), and the hJ_λ segment was associated with a 12-RSS in reverse orientation. The mouse V_κ cluster retains its endogenous 3' terminal 12-RSS. Based on the 12-23 rule of V(D)J recombination, the 12-RSS hJ_λ segment can only recombine with the 23-RSS hV_λ, but not the 12-RSS mouse V_κ segments, which lie upstream of the cassette. Consequently, the humanized Igk allele should express exclusively human variable regions, despite the presence of intact mV_κ cluster. Mouse C_κ (mC_k) was replaced with human C_λ (IGLC3*04).

Embryonic stem cells carrying the Igk mutant allele were re-targeted to introduce a CAG-hTdT cassette to intron 1 of the mouse Gt(ROSA)26Sor gene, downstream of the Igk allele on Chromosome 6. Expression of the human terminal deoxynucleotidyl transferase (hTdT; formal gene symbol DNTT, DNA nucleotidylexotransferase) cDNA is driven by the CAG promotor during light chain rearrangement in pre-B cells. It adds random nucleotides to V_L and J_Ljunctions to form N regions, and promotes human-like diversification of the light chain cassette.

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 (IGHV3-33*05); 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^tm4.1Fwa
Name: targeted mutation 4.1, Frederick W Alt
MGI accession ID: MGI:8394050
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: germline human V (hVλ; IGLV2-14*01) was incorporated in the 3' region of the mouse Vκ (mVk) cluster and J (hJλ; IGLJ3*01) replaces all five mouse Jκ (mJk) segments. The hVλ segment was tagged with a recombination signal sequence incorporating a 23-base pair spacer (23-RSS), and the hJλ segment was associated with a 12-RSS in reverse orientation. The mouse Vκ cluster retains its endogenous 3' terminal 12-RSS. Based on the 12-23 rule of V(D)J recombination, the 12-RSS hJλ segment can only recombine with the 23-RSS hVλ, but not the 12-RSS mouse Vκ segments, which lie upstream of the cassette. Consequently, the humanized Igk allele should express exclusively human variable regions, despite the presence of intact mVκ cluster. Mouse Cκ (mCk) was replaced with human Cλ (IGLC3*04). 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^tm15.1Fwa
Name: targeted mutation 15.1, Frederick W Alt
MGI accession ID: MGI:8394053
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 (IGHV3-33*05); 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.

Allele

Symbol: Gt(ROSA)26Sor^{tm4(CAG-DNTT)Fwa}
Name: targeted mutation 4, Frederick W Alt
MGI accession ID: MGI:8394051
Generation method: Targeted
Attributes: Inserted expressed sequence
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: 129S/SvEv x C57BL/6
Molecular note: Embryonic stem cells carrying the Igk mutant Igk^tm4.1Fwa allele were re-targeted to introduce a CAG-hTdT cassette to intron 1 of the mouse Gt(ROSA)26Sor gene, downstream of the Igk allele on Chromosome 6. Expression of the human terminal deoxynucleotidyl transferase (hTdT; formal gene symbol DNTT, DNA nucleotidylexotransferase) cDNA is driven by the CAG promotor during light chain rearrangement in pre-B cells. It adds random nucleotides to VH-D and D-JH junctions to form N regions, and promotes human-like diversification of the light chain cassette.