A simple and rapid protocol for the sequence determination of functional kappa light chain cDNAs from aberrant-chain-positive murine hybridomas.
This protocol describes the application of a polymerase chain reaction to allow the cloning and sequencing of new functional kappa light chain cDNAs from murine hybridomas co-expressing aberrant endogenous kappa chain mRNAs. The presence of kappa light chain aberrant mRNAs can hinder or even prevent determination of the sequence of functional murine kappa light chain cDNAs amplified by PCR from hybridomas. The method described here employs a panel of kappa primers in the presence of molar excess of a primer complementary to the complementary determining region (CDR) 3 of the known aberrant chain sequence. Analysis of the PCR products reveals two bands for some reactions: one the functional, full-length kappa chain cDNA (approximately 400 bp) and another shorter (approximately 100 bp) band corresponding to short aberrant chain kappa CDR3-constant region. The full-length product is gel purified and cloned prior to sequencing and aligned with V-region germline sequences available in NCBI and GenBank databases. This method is used routinely in our laboratory and demonstrates consistency and reliability for sequence determination of kappa light chain V-gene cDNA of mAbs to diverse antigens. This protocol is a rapid and convenient method for determining the sequence of murine V kappa region genes from hybridomas expressing aberrant kappa chain mRNAs.
Yuan
Xin
X
The Monoclonal Antibody Section, National Microbiology Laboratory-Health Canada and the National Centre for Foreign Animal Disease-CFIA, 1015 Arlington Street, Winnipeg, Manitoba, Canada.
Gubbins
Michael J
MJ
Berry
Jody D
JD
D000595:Amino Acid Sequence / Q000235:genetics*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D030561:Databases, Nucleic Acid; D005786:Gene Expression Regulation; D015328:Gene Rearrangement, B-Lymphocyte, Light Chain / Q000235:genetics*; D006825:Hybridomas; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D017384:Sequence Deletion / Q000235:genetics*
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D017931:DNA Primers; D018076:DNA, Complementary; D007145:Immunoglobulin kappa-Chains; D012333:RNA, Messenger