Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction

Weissenhorn, Winfried; Weiss, Elisabeth H.; Schwirzke, Marina; Kaluzab, Brigitte und Weidle, Ulrich H. (1991): Chimerization of antibodies by isolation of rearranged genomic variable regions by the polymerase chain reaction. In: Gene, Bd. 106: S. 273-277

Abstract

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 ofAbs. A fundamental prerequisite for this is the knowledge ofthe 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.

• Dokumententyp: Zeitschriftenartikel
• Keywords: Hybridoma; transfectoma; force cloning; cassette vectors; PCR; recombinant DNA
• Fakultät: Biologie
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
• Sprache: Englisch