B6;129-Igk^em2Abtx/Igk^{em4(IGK*)Abtx} Igh^{em1(IGH*)Abtx}/Igh^em2Abtx/J

Stock number: 039319
Product name: Atlas™ Binary Fixed Light Chain Mouse
Strain synonyms: KLaP (k-KO) ; Fixed LC :: Fully human HC ; mIgDJ_D KO
Research areas: Cancer Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

The Atlas™ Binary Fixed Light Chain Mouse is a platform for generating diverse human variable region and mouse constant region monoclonal antibodies upon immunization with a constant light chain. This model enables target-driven expression of one of two fixed light chains, each with overlapping yet distinct abilities to pair with cognate variable heavy chains, thereby facilitating the development of a bispecific antibody format similar to native antibodies.

Customers must have the appropriate agreement in place to purchase this strain.  For more information, please contact Atlas@jax.org.

The Atlas™ Full Human Diversity Mouse for the generation of chimeric monoclonal antibodies is also available as Stock No. 039317.

Detailed description

Atlas™ Binary Fixed Light Chain Mice were created by targeted knock-ins of the human V(D)J heavy regions and two fixed VJ kappa light regions, replacing the endogenous variable regions of the murine immunoglobulin loci. This model allows for target-driven expression from one of two fixed light chains which contain overlapping yet distinct abilities to pair with cognate variable heavy chains. The selection of the cognate fixed light chain in this model is based on optimal pairing during the B cell maturation process in response to the specific antigen presented. Obtaining antibody leads with common light chains facilitates bringing both antibody arms together post discovery to create bispecific antibodies without linkers, fusions, or modifications.

By containing the near full complement of human heavy chain antibody gene sequence diversity with robust antibody titers compared to wildtype mice, Atlas™ Mouse antibodies are selected for and matured in vivo, simplifying downstream engineering and streamlining the discovery and development process.

Key Benefits:

• Variable regions comprising >99% of commonly expressed human heavy chain sequences reduce the risk of immunogenicity and improve antibody developability characteristics by limiting the need for downstream engineering.
• Knock-in to endogenous mouse loci promotes physiologically-relevant expression and generation of antibody sequences.
• Dual light chain optionality increases hit frequency and diversity compared to single fixed light chain variants.
• Common light chain format allows creation of natural, paired antibody structures for improved pharmacokinetics, tolerability, and manufacturability.

Development

The Atlas™ Binary Fixed Light Chain Mouse (Stock No. 039319) is distributed as F1 (first filial generation) mice resulting from breeding the double homozygous KLaP (k-KO) :: mIgDJ_D KO strain (B6;129-Igk^em2Abtx Igh^em2Abtx/J) with the double homozygous Fixed LC :: Fully human HC strain (B6;129-Igk^{em4(IGK*)Abtx} Igh^{em1(IGH*)Abtx}/J). The resulting F1 mice are compound heterozygous at the Igk and Igh loci - each of the four mutations is described below.

The KLaP (k-KO) allele (Igk^em2Abtx) has the mouse immunoglobulin kappa chain complex (Igk) region variable (V) and joining (J) segments deleted. Specifically, CRISPR/Cas9 genome editing in 129S6/SvEvTac-derived CMTI-1 embryonic stem (ES) cells was used to remove a ~3.2 Mb sequence including the mouse Igk V and J segments, and replace it with a PGK-neomycin resistance cassette. The transcript ID / genome build used as a reference for the sequence coordinates or guide/donor sequences were not provided. The mouse Igk 3' enhancer and constant regions remain telomeric to the insertion. Sequence confirmed founders were bred to C57BL/6N mice for germline transmission, and later IVF-expanded using C57BL/6J female donors. The resulting mice were maintained on a mixed C57BL/6N;C57BL/6J;129S6 genetic background before being bred with the C57BL/6N;C57BL/6J;129S6 mIgDJ_D KO strain.

The mIgDJ_D KO allele (Igh^em2Abtx) has the mouse immunoglobulin heavy chain complex (Igh) region diversity (D) and joining (J) segments deleted. Specifically, CRISPR/Cas9 genome editing in 129S6/SvEvTac-derived CMTI-1 embryonic stem (ES) cells was used to remove a ~55 kb mouse Igh D-J segment and replace it with a PGK-neomycin resistance cassette. The transcript ID / genome build used as a reference for the sequence coordinates or guide/donor sequences were not provided. The mouse Adam6b/Adam6a loci remain telomeric to the deleted region, and the mouse Igh heavy chain constant regions (μ, δ, γ, ε, α) remain centromeric to the deleted region. Sequence confirmed founders were bred to C57BL/6N mice for germline transmission, and later IVF-expanded using C57BL/6J female donors. The resulting mice were maintained on a mixed C57BL/6N;C57BL/6J;129S6 genetic background before being bred with the C57BL/6N;C57BL/6J;129S6 KLaP (k-KO) strain.

The Fixed LC allele (Igk^{em4(IGK*)Abtx}) has the mouse immunoglobulin kappa chain complex (Igk) region variable (V), joining (J), MAR/intronic enhancer (iE) and constant (C) segments deleted, then replaced with a human IGK 'fixed light chain' and C region sequence. Specifically, CRISPR/Cas9 genome editing in 129S6/SvEvTac-derived CMTI-1 embryonic stem (ES) cells was used to remove a ~3.2 Mb sequence including the mouse Igk V-J-C segments and replace it with a ~2 kb 'landing pad' comprised of a loxP-flanked neomycin resistance cassette (Igk^em3Abtx). A subsequent recombinase-mediated cassette exchange step was carried out with Cre recombinase to replace the floxed-neo with a loxP-flanked ~19.6 kb sequence encoding a human IGK synthetic 'fixed light chain' sequence followed by the human genomic IGK C sequence. The 'fixed light chain' sequence is comprised of two pre-rearranged human IGK V-J segments primarily surrounded by endogenous 5' and 3' flanking DNA sequences. This sequence is inactive in the germline state. As such, the sequence was further modified to include Recombination Signal Sequence (RSS) motifs to facilitate downstream B-cell specific recombination and reconstitution of functional transcripts for one or the other IGK V-J cassette, in a stochastic fashion. The transcript ID / genome build used as a reference for the sequence coordinates or guide/donor sequences were not provided. The allele also retains a puromycin resistance cassette centromeric to the insertion. Sequence confirmed Fixed LC allele founders (Igk^{em4(IGK*)Abtx}) were bred to C57BL/6N mice for germline transmission, and later IVF-expanded using C57BL/6J female donors. The resulting mice were maintained on a mixed C57BL/6N;C57BL/6J;129S6 genetic background before being bred with the C57BL/6N;C57BL/6J;129S6 Fully human HC strain.

The Fully human HC allele (Igh^{em1(IGH*)Abtx}) has the mouse immunoglobulin heavy chain complex (Igh) region variable (V), diversity (D) and joining (J) segments deleted and replaced with a human IGH V-D-J sequence. Specifically, CRISPR/Cas9 genome editing in 129S6/SvEvTac-derived CMTI-1 embryonic stem (ES) cells was used in two separate steps to deliver into the mouse Igh locus a ~139 kb sequence containing an array of 41 IGH V segments plus a fully functional complement of 27 IGH D and 6 IGH J gene segments. The transcript ID / genome build used as reference for the sequence coordinates or guide/donor sequences were not provided. The mouse Adam6b/Adam6a loci remain telomeric to the human insertion, and the mouse Igh heavy chain constant regions (μ, δ, γ, ε, α) remain centromeric to the insertion. The allele also retains both puromycin resistance and hygromycin resistance cassettes telomeric of the insertion. Sequence confirmed founders were bred to C57BL/6N mice for germline transmission, and later IVF-expanded using C57BL/6J female donors. The resulting mice were maintained on a mixed C57BL/6N;C57BL/6J;129S6 genetic background before being bred with the C57BL/6N;C57BL/6J;129S6 Fully-Hu KLC strain or the C57BL/6N;C57BL/6J;129S6 Fixed LC strain.
Of note, the hygromycin resistance cassette sequence present in the Fully human HC allele is unique and does not amplify using The Jackson Laboratory's current Generic Hygro assay primers [August 2024].

Allele

Symbol: Igk^em2Abtx
Name: endonuclease-mediated mutation 2, Dan Rohrer
MGI accession ID: MGI:7715232
Generation method: Endonuclease-mediated
Attributes: Null/Knockout
Synonyms: KLaP (k-KO)
Marker symbol: Igk
Marker name: immunoglobulin kappa chain complex
Marker synonyms: kappa
Chromosome: 6
Strain of origin: 129S6/SvEvTac
Molecular note: The allele has the mouse immunoglobulin kappa chain complex (Igk) region variable (V) and joining (J) segments deleted. Specifically, CRISPR/Cas9 genome editing in 129S6/SvEvTac-derived CMTI-1 embryonic stem (ES) cells was used to remove a ~3.2 Mb sequence including the mouse Igk V and J segments and replace it with a PGK-neomycin resistance cassette.

Allele

Symbol: Igk^{em4(IGK*)Abtx}
Name: endonuclease-mediated mutation 4, Dan Rohrer
MGI accession ID: MGI:7715234
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: Fixed LC
Marker symbol: Igk
Marker name: immunoglobulin kappa chain complex
Marker synonyms: kappa
Chromosome: 6
Strain of origin: 129S6/SvEvTac
Molecular note: The allele has the mouse immunoglobulin kappa chain complex (Igk) region variable (V), joining (J), MAR/intronic enhancer (iE) and constant (C) segments deleted, then replaced with a human IGK 'fixed light chain' and C region sequence. Specifically, CRISPR/Cas9 genome editing in 129S6/SvEvTac-derived CMTI-1 embryonic stem (ES) cells was used to remove a ~3.2 Mb sequence including the mouse Igk V-J-C segments and replace it with a ~2 kb 'landing pad' comprised of a loxP-flanked neomycin resistance cassette. Subsequently this Igk^em3Abtx allele was used for a recombinase-mediated cassette exchange (RMCE) step with Cre recombinase to replace the floxed-neo with a loxP-flanked ~19.6 kb sequence encoding a human IGK synthetic 'fixed light chain' sequence followed by the human genomic IGK C sequence. The 'fixed light chain' sequence is comprised of two pre-rearranged human IGK V-J segments primarily surrounded by endogenous 5' and 3' flanking DNA sequences. This sequence is inactive in the germline state. As such, the sequence was further modified to include Recombination Signal Sequence (RSS) motifs to facilitate downstream B-cell specific recombination and reconstitution of functional transcripts for one or the other IGK V-J cassette, in a stochastic fashion.

Allele

Symbol: Igh^{em1(IGH*)Abtx}
Name: endonuclease-mediated mutation 1, Dan Rohrer
MGI accession ID: MGI:7715238
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: Fully human HC
Marker symbol: Igh
Marker name: immunoglobulin heavy chain complex
Chromosome: 12
Strain of origin: 129S6/SvEvTac
Molecular note: The allele has the mouse immunoglobulin heavy chain complex (Igh) region variable (V), diversity (D) and joining (J) segments deleted and replaced with a human IGH V-D-J sequence. Specifically, CRISPR/Cas9 genome editing in 129S6/SvEvTac-derived CMTI-1 embryonic stem (ES) cells was used in two separate steps to deliver into the mouse Igh locus a ~139 kb sequence containing an array of 41 IGH V segments plus a fully functional complement of 27 IGH D and 6 IGH J gene segments.

Allele

Symbol: Igh^em2Abtx
Name: endonuclease-mediated mutation 2, Dan Rohrer
MGI accession ID: MGI:7715239
Generation method: Endonuclease-mediated
Attributes: Null/Knockout
Synonyms: mIgDJ_D KO
Marker symbol: Igh
Marker name: immunoglobulin heavy chain complex
Chromosome: 12
Strain of origin: 129S6/SvEvTac
Molecular note: The allele has the mouse immunoglobulin heavy chain complex (Igh) region diversity (D) and joining (J) segments deleted. Specifically, CRISPR/Cas9 genome editing in 129S6/SvEvTac-derived CMTI-1 embryonic stem (ES) cells was used to remove a ~55 kb mouse Igh D-J segment and replace it with a PGK-neomycin resistance cassette.