Rapid isolation of immunoglobulin variable genes from cell lysates of rat hybridomas by polymerase chain reaction.
The isolation of the rearranged immunoglobulin genes from a hybridoma cell line, which is a prerequisite for the construction of a recombinant antibody, can easily be achieved by polymerase chain reaction. Here we demonstrate that this method, which was originally described for cloning murine immunoglobulin genes from cDNA, is also applicable for rat genes. We show that the procedure also works with crude cell lysates as starting material, thereby greatly reducing the time required for sample preparation. In addition we have sequenced the nonfunctional heavy chain variable gene of the fusion partner X63Ag8.653, which was readily amplified from our hybridoma cells, and whose sequence has been so far unknown.
Kütemeier
G
G
GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Immunologie, München, Germany.
Harloff
C
C
Mocikat
R
R
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006825:Hybridomas / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D019914:Taq Polymerase
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase