Transcription from plasmid expression vectors is increased up to 14-fold when plasmids are transfected as concatemers.
A protocol for increasing transcription from plasmid expression vectors is presented. A vector containing chloramphenicol acetyltransferase (CAT) gene was digested leaving the transcription cassette intact. Heat inactivation of restriction enzymes followed by ligation of the digestion products yielded concatemers which migrated as a single band in agarose gel electrophoresis. Mouse fibroblasts transfected with the concatemers gave a CAT activity that was 14-fold greater than that of cells transfected with a similar mass (equimolar gene number) of the native plasmid. The effect was independent of promoter type, restriction enzyme, number of restriction sites and with a noted exception, cell line.
Leahy
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Department of BioStructure and Function, University of Connecticut Health Center, Farmington, CT 06030, USA. px124@po.cwru.edu
Carmichael
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GG
Rossomando
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EF
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005347:Fibroblasts; D015870:Gene Expression / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection / Q000235:genetics*
D015500:Chloramphenicol O-Acetyltransferase; D004262:DNA Restriction Enzymes