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Nat Commun. 2016 Aug 17;7:12535. doi: 10.1038/ncomms12535.

Immunogenomic engineering of a plug-and-(dis)play hybridoma platform.

Author information

1
Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, Basel 4058, Switzerland.
2
Life Science Zurich Graduate School, Systems Biology, ETH Zurich, University of Zurich, Zurich 8057, Switzerland.

Abstract

Hybridomas, fusions of primary mouse B cells and myelomas, are stable, rapidly-proliferating cell lines widely utilized for antibody screening and production. Antibody specificity of a hybridoma clone is determined by the immunoglobulin sequence of the primary B cell. Here we report a platform for rapid reprogramming of hybridoma antibody specificity by immunogenomic engineering. Here we use CRISPR-Cas9 to generate double-stranded breaks in immunoglobulin loci, enabling deletion of the native variable light chain and replacement of the endogenous variable heavy chain with a fluorescent reporter protein (mRuby). New antibody genes are introduced by Cas9-targeting of mRuby for replacement with a donor construct encoding a light chain and a variable heavy chain, resulting in full-length antibody expression. Since hybridomas surface express and secrete antibodies, reprogrammed cells are isolated using flow cytometry and cell culture supernatant is used for antibody production. Plug-and-(dis)play hybridomas can be reprogrammed with only a single transfection and screening step.

PMID:
27531490
PMCID:
PMC4992066
DOI:
10.1038/ncomms12535
[Indexed for MEDLINE]
Free PMC Article

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