In combination with immunoglobulin lambda light chain, this heavy chain variable (V) region produces antibody that binds hapten NP (4-hydroxy-3-nitrophenylacetyl). An Ala to Gly point mutation at codon 24, Ser to Thr point mutation at codon 31, and a His to Gln point mutation at codon 35 result in low binding affinity for antigen (B1-8lo). Despite the significantly enhanced affinity for NP, there is only a 2-3 fold difference in the T-independent proliferative response of B cells that carry high or low affinity receptors. In T cell-dependent responses there is no difference in the ability of B1-8 high vs. B1-8 low affinity cells to enter germinal center reactions. However, high affinity B cells were recruited preferentially in response to both T cell-dependent and T cell-independent antigens.
Michel C Nussenzweig, The Rockefeller University
Mice homozygous for this targeted mutation are viable and fertile and do not display any gross physical or behavioral abnormalities. In combination with immunoglobulin lambda light chain, this heavy chain variable (V) region produces antibody that binds hapten NP (4-hydroxy-3-nitrophenylacetyl). An Ala to Gly point mutation at codon 24, Ser to Thr point mutation at codon 31, and a His to Gln point mutation at codon 35 result in low binding affinity for antigen (B1-8lo). Despite the significantly enhanced affinity for NP, there is only a 2-3 fold difference in the T-independent proliferative response of B cells that carry high or low affinity receptors. In T cell-dependent responses there is no difference in the ability of B1-8 high vs. B1-8 low affinity cells to enter germinal center reactions. However, high affinity B cells were recruited preferentially in response to both T cell-dependent and T cell-independent antigens. Expression has been verified by immunostaining cells that carry the mutation.
PCR was used to introduce three point mutations conferring lower affinity 4-hydroxy-3-nitrophenyl)acetyl (NP)-binding to Igh (VHB1-8): GCT->GGT (codon 24 Ala->Gly), AGC->ACC (codon 31 Ser->Thr), CAC-CAG (codon 35 His->Gln). A targeting vector incorporating a 5' floxed neomycin cassette in addition to the point mutations was transfected into 129P2/OlaHsd-derived E14 embryonic stem (ES) cells. Positive clones were injected into C57BL/6 blastocysts, and the resulting chimeric males were bred to C57BL/6 EIIA-Cre females to delete the neomycin gene, and leave a loxP site. This strain has been backcrossed to C57BL/6 for more than 12 generations by the donating laboratory.
|Allele Name||targeted mutation 2, Michel C Nussenzweig|
|Gene Symbol and Name||Igh, immunoglobulin heavy chain complex|
|Promoter||Igh, immunoglobulin heavy chain complex, mouse, laboratory|
|Strain of Origin||129P2/OlaHsd|
|Molecular Note||The following point mutations were inserted into the VHB1-8 region: GCT to GGT (Ala24Gly), AGC to ACC (Ser31Thr), CAC to CAG (His35Gln), AGA to ACT (Arg98Thr). A floxed neo included in the vector was subsequently removed via cre mediated recombination.|
|Mutations Made By|| |
Michel Nussenzweig, The Rockefeller University
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