Cloning and expression of murine Ig genes from single B cells.
We have established a highly efficient 96-well format based strategy to characterize the expressed murine antibody repertoire by combining immunoglobulin (Ig) gene cloning with antibody expression and reactivity profiling at the single cell level. Individual mouse B lineage cells are isolated based on defined surface marker expression patterns by fluorescence-activated cell sorting (FACS) and corresponding full-length Ig heavy (H) and Ig light (L) chain variable (V) region gene transcripts are amplified by RT-PCR. Cloning of the amplified products into eukaryotic expression vectors enables the in vitro production of monoclonal antibodies with antigen specificities identical to the initial B cell antigen receptors. IgH and IgL chain gene sequence information is obtained as part of the cloning procedure and can be directly linked to reactivity profiles of the recombinant antibodies. In summary, our RT-PCR based strategy to generate recombinant monoclonal antibodies from single mouse B cells allows the highly efficient and unbiased characterization of the expressed murine antibody repertoire by sequence analysis and parallel antibody reactivity testing.
Tiller
Thomas
T
Max Planck Institute for Infection Biology, Charitéplatz 1, Berlin D-10117, Germany. tiller@mpiib-berlin.mpg.de
Busse
Christian E
CE
Wardemann
Hedda
H
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region