Expression of immunoglobulin genes tandem in eukaryotic cells under the control of T7 bacteriophage RNA polymerase.
A tandem of recombinant mouse/human immunoglobulin (Ig) genes was constructed and inserted into the plasmid pGEM1 under the control of T7 phage RNA polymerase promoter. Sp2/0 lymphoid cell line and Chinese Hamster Ovary (CHO) cells were used as the targets for gene transfection. Both cell lines contained in their genomes a T7 RNA polymerase gene modified with a nuclear-located signal derived from SV40 large T-antigen. Cell lines transfected with the gene tandem effectively synthesized mRNA (up to 9 x 10(3) bp) that hybridized with epsilon- and kappa-gene probes. Separate transcripts corresponding to mRNAs of individual heavy and light chains were not detected in either transfected cell line. It follows from these data that transcription in the transfected cells is controlled mainly by the T7 phage polymerase promoter. Both lymphoid and nonlymphoid cell lines transfected with the gene tandem synthesized the epsilon-heavy (70 kDa) and kappa-light (25 kDa) Ig polypeptide chains. Production of chimeric antibodies by the myeloma Sp2/0 cells was higher than that by the CHO cells. Individual clones synthesized up to 150 ng/mL chimeric IgE. However, only lymphoid Sp2/0 cells were capable of efficient secretion of the recombinant antibodies. The mechanism of translation of mRNA synthesized in eukaryotic cells by T7 phage RNA polymerase is discussed.[1]References
- Expression of immunoglobulin genes tandem in eukaryotic cells under the control of T7 bacteriophage RNA polymerase. Deyev, S.M., Lieber, A., Radko, B.V., Polanovsky, O.L. Appl. Biochem. Biotechnol. (1994) [Pubmed]
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