Specific amplification of rearranged immunoglobulin variable region genes from mouse hybridoma cells.
In this article we show how the polymerase chain reaction (PCR) and primers designed for conserved sequences of leader (L), framework one (FR1) and constant (CONST) regions of immunoglobulin light and heavy chain genes can be used for the cloning and sequencing of rearranged antibody variable regions from mouse hybridoma cells. RNA was extracted from the mouse hybridoma cells secreting MAbs: IOR-T3a (anti-CD3), C6 (anti-P1 of N. meningitidis B385), IOR-T1 (anti-CD6), CB-CEA.1 (anti-carcinoembryonic antigen), and CB-Fib.1 (anti-human fibrin). First strand cDNA was synthesized and amplified using PCR. The newly designed primers are superior to others reported recently in the literature. Isolated PCR DNA fragments of C6 and IOR-T3a were sequenced after asymmetric amplification, or M13 cloning. The FR1/CONST primer combinations selectively amplified mouse lights chain of groups kappa II, V, and VI, and heavy chains of groups IIa and IIc. The L/CONST primers for light chains amplified light chains from all four hybridomas. These methods greatly facilitate structural and functional studies of antibodies by reducing the efforts required to clone and sequence their variable regions.
Gavilondo-Cowley
J V
JV
Division of Hybridomas and Animal Models, Center for Genetic Engineering and Biotechnology, La Habana, Cuba.
Coloma
M J
MJ
Vazquez
J
J
Ayala
M
M
Macías
A
A
Fry
K E
KE
Larrick
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005784:Gene Amplification; D015321:Gene Rearrangement; D005803:Genes, Immunoglobulin; D006825:Hybridomas / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D007135:Immunoglobulin Variable Region; D004247:DNA