Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction.
We describe a new method for amplification, by polymerase chain reaction (PCR), of rearranged segments encoding the variable part of light and heavy chains of an antibody (Ab) from the chromosomal DNA of hybridoma cells for the chimerization of Abs. A fundamental prerequisite for this is the knowledge of the exact sequences in the 5'-untranslated region of light and heavy chain mRNA, and of the joining segment used for rearrangement. This allows the design of nondegenerated oligodeoxyribonucleotides for PCR. The primer design permits directional cloning of the amplified, promoterless fragments into cassette vectors, in which they will be linked to the appropriate human constant domains and immunoglobulin (Ig) promoter/enhancer elements. The method is illustrated for chimerization of an Ab directed against the human T-lymphocyte antigen, CD4. The chimerized Ab is secreted in abundant quantities after transfection of the engineered plasmids into non-Ig-producing myeloma cells.
Institut für Immunologie, Universität München, F.R.G.
W
W
Weissenhorn
E
E
Weiss
M
M
Schwirzke
B
B
Kaluza
U H
UH
Weidle
D000818:Animals; D001483:Base Sequence; D015704:CD4 Antigens / Q000276:immunology; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015321:Gene Rearrangement / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D006825:Hybridomas; D007133:Immunoglobulin Joining Region / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D007146:Immunoglobulin lambda-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D015704:CD4 Antigens; D004274:DNA, Recombinant; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D009838:Oligodeoxyribonucleotides