title: Sequencing and cloning of antigen-specific antibodies from mouse memory B cells.
abstract: Methods to identify genes encoding immunoglobulin heavy and light chains from single B lymphocytes vary in efficiency, error rate and practicability. Here we describe a protocol to sequence and clone the variable antibody region of single antigen-specific mouse memory B cells for antibody production. After purification, antigen-specific mouse memory B cells are first single-cell-sorted by fluorescence-activated cell sorting (FACS), and V(D)J transcripts are amplified by RT-PCR. Fragments are then combined with linearized expression vectors, assembled in vitro as part of a sequence- and ligation-independent cloning (SLIC) reaction and then transformed into Escherichia coli. Purified vectors can then be used to produce monoclonal antibodies in HEK293E suspension cells. This protocol improves the amplification efficiency of antibody variable genes and accelerates the cloning workflow. Antibody sequences will be available in 3-4 d, and microgram to milligram amounts of antibodies are produced within 14 d. The new protocol should be useful for addressing fundamental questions about antigen-specific memory B cell responses, as well as for characterizing antigen-specific antibodies.
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.
authors.forename: Lotta
authors.initials: L
authors.lastname: von Boehmer
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.
authors.forename: Cassie
authors.initials: C
authors.lastname: Liu
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Ackerman
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Gitlin
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.|Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Gazumyan
authors.affiliation: Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.|Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Michel C
authors.initials: MC
authors.lastname: Nussenzweig