Restriction-PCR fingerprinting of the immunoglobulin VH repertoire: direct detection of an immune response and global analysis of B cell clonality

Abstract

Here we describe a method for fingerprinting the mouse immunoglobulin heavy chain variable region (VH) repertoire. Using a novel combination of existing techniques, large numbers of expressed VH genes can be simultaneously displayed as a fingerprint of VH gene fragments on a sequencing gel. This is achieved using isotype-specific reverse transcription-PCR amplification, restriction digestion and end-labeled primer runoffs. This technique (Res-PCR) allows analysis of the immune response, in this case to phenyloxazolone, in several different tissues and enables molecular cloning and sequencing of the VH genes involved in vivo. In addition, with Res-PCR, a "global" picture of expressed immunoglobulin genes is represented. This elucidates B cell clonality in different tissues and isotypes and detects a preference for shorter complementarity-determining region 3 in IgM-expressing cells. Res-PCR is a rapid and revealing method which will allow analysis of the complexity and sequence composition of the B cell immunoglobulin repertoire and so perhaps better define the B cell memory compartment and immune responses in vivo.

Generation of a poor prognostic chronic lymphocytic leukemia-like disease model: PKC subversion induces up-regulation of PKC II expression in B lymphocytes

“…cDNA was amplified with PCR primer combinations and cycles described elsewhere. 15 Successfully amplified PCR products were cloned into pCRII-Blunt-TOPO (Invitrogen) and sequenced with M13 reverse/forward primers. The data acquired were analyzed using IMGT (www.imgt.org).…”

Intrinsic defects of B cell function in X-linked severe combined immunodeficiency

“…To detect the presence of class-switched Ig expression and clonal expansions in peripheral blood B cells from X-SCID patients and normal donors, RT-PCR analysis using Ig VH1 and H chain subclass-specific primers was performed. Under such circumstances a ladder of transcript sizes is detected, representing the in-frame distribution of Ig lengths created by VDJ recombination [22].…”

“…However, the evidence for this has to date come from in vitro studies and indirectly from the transplantation studies above. We have used a reverse transcription (RT)-PCR-based technique to study the isotype and diversity of the expressed Ig repertoire [22]. This assay allows us to look at the ability of host + c-defective and normal donor B cells to undergo class switching and clonal expansion pre and post HSCT.…”

CD21intCD23+ follicular B cells express antigen‐specific secretory IgM mRNA as primary and memory responses

“…The hapten-carrier phOx-CSA was made as described previously. 28,29 For primary immunizations (day 0) 100 lg of alum-precipitated phOx-CSA with 10 9 heatkilled pertussis was injected intraperitoneally. For boosting (day 50) 100 lg soluble phOx-CSA in PBS was injected iintraperitoneally.…”

“…The phOx/RT-PCR method is highly reproducible, detecting up-regulated IgM and/or IgG transcripts in every immunized mouse of the appropriate IgH haplotype. 14,24,28 Further, for reasons dealt with in depth elsewhere, 14 the RT-PCR method used is independent of known mechanisms of PCR bias.…”