Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR.
To facilitate the rapid cloning and sequencing of rearranged murine heavy-chain variable regions, we have designed a set of universal primers using conserved sequences of leader (signal peptide), framework one and constant regions of the immunoglobulin heavy-chain genes. RNA was extracted from the mouse hybridoma cells secreting monoclonal antibodies: IOR-T3 (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), CB-Fib.1 (anti-human fibrin) and CB-Hep.2 (anti-hepatitis B surface antigen). First-strand cDNA was synthesized and amplified using PCR. The primers successfully amplified correct size fragments from cDNA prepared from all hybridomas. These methods will facilitate the cloning and sequencing of mouse immunoglobulin variable regions.
Coloma
M J
MJ
Genelabs, Inc., Redwood City, CA 94063.
Larrick
J W
JW
Ayala
M
M
Gavilondo-Cowley
J V
JV
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011089:Polydeoxyribonucleotides; D004247:DNA