High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.
Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.
D000123:Acetyltransferases / Q000235:genetics*; D000818:Animals; D002130:Calcium Phosphates / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase; D003637:DEAE-Dextran / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology; D007739:L Cells; D051379:Mice; D014162:Transfection / Q000187:drug effects*
D002130:Calcium Phosphates; D003637:DEAE-Dextran; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D005990:Glycerol; D004121:Dimethyl Sulfoxide