Recombinase-Mediated Cassette Exchange as a Novel Method To Study Somatic Hypermutation in Ramos Cells
| Autor: | Manxia Fan, Linda B. Baughn, Aviv Bergman, Lirong Wei, Susan L. Kalis, Matthew D. Scharff, Thomas MacCarthy |
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| Rok vydání: | 2011 |
| Předmět: |
Molecular Sequence Data
Immunoglobulin Variable Region Somatic hypermutation Microbiology Recombinases Affinity maturation 03 medical and health sciences 0302 clinical medicine Mutation Rate Cell Line Tumor Virology medicine Humans B cell 030304 developmental biology Recombination Genetic Genetics 0303 health sciences Base Sequence biology Recombinase-mediated cassette exchange Germinal center Antibody Diversity Cytidine deaminase Burkitt Lymphoma QR1-502 3. Good health Cell biology medicine.anatomical_structure Genetic Techniques biology.protein Somatic Hypermutation Immunoglobulin Antibody Immunoglobulin Heavy Chains Research Article 030215 immunology |
| Zdroj: | mBio mBio, Vol 2, Iss 5 (2011) |
| ISSN: | 2150-7511 2161-2129 |
| DOI: | 10.1128/mbio.00186-11 |
| Popis: | Activation-induced cytidine deaminase (AID) mediates the somatic hypermutation (SHM) of immunoglobulin (Ig) variable (V) regions that is required for the generation of antibody diversity and for the affinity maturation of the antibody response against infectious agents and toxic substances. AID preferentially targets WRC (W = A/T, R = A/G) hot spot motifs, particularly WGCW motifs that create overlapping hot spots on both strands. In order to gain a better understanding of the generation of antibody diversity and to create a platform for the in vitro generation of affinity-matured antibodies, we have established a system involving recombinase-mediated cassette exchange (RMCE) to replace the V region and its flanking sequences. This makes it possible to easily manipulate the sequence of the Ig gene within the endogenous heavy chain of the Ramos human Burkitt’s lymphoma cell line. Here we show that the newly integrated wild-type (WT) VH regions introduced by RMCE undergo SHM similarly to non-RMCE-modified Ramos cells. Most importantly, we have shown that introducing a cluster of WGCW motifs into the complementary determining region 2 (CDR2) of the human heavy chain V region significantly raised the mutation frequency and number of mutations per sequence compared to WT controls. Thus, we have demonstrated a novel platform in Ramos cells whereby we can easily and quickly manipulate the endogenous human VH region to further explore the regulation and targeting of SHM. This platform will be useful for generating human antibodies with changes in affinity and specificity in vitro. IMPORTANCE An effective immune response requires a highly diverse repertoire of affinity-matured antibodies. Activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) of immunoglobulin (Ig) genes. Although a great deal has been learned about the regulation of AID, it remains unclear how it is preferentially targeted to particular motifs, to certain locations within the Ig gene and not to other highly expressed genes in the germinal center B cell. This is an important question because AID is highly mutagenic and is sometimes mistargeted to other highly expressed genes, including proto-oncogenes, leading to B cell lymphomas. Here we describe how we utilize recombinase-mediated cassette exchange (RMCE) to modify the sequence of the endogenous heavy chain locus in the Ramos Burkitt’s lymphoma cell line. This platform can be used to explore the regulation and targeting of SHM and to generate human antibodies with changes in affinity and specificity in vitro. |
| Databáze: | OpenAIRE |
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