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

Margaret A. Lopata, Don W. Cleveland, Barbara Sollner-webb

Research output: Contribution to journalArticle

Abstract

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 trsnafection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expreasion of the transfected gene a surprising ̃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 aulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextrsn coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.

Original languageEnglish (US)
Pages (from-to)5707-5717
Number of pages11
JournalNucleic Acids Research
Volume12
Issue number14
DOIs
StatePublished - Jul 25 1984

Fingerprint

DEAE-Dextran
Dextran
Dimethyl sulfoxide
Chloramphenicol
Transferases
Dimethyl Sulfoxide
Glycerol
Transfection
Shock
DNA
Genes
Gene
Plasmids
Cell
High Efficiency
Mouse
Fold
Inclusion

ASJC Scopus subject areas

  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)
  • Genetics

Cite this

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. / Lopata, Margaret A.; Cleveland, Don W.; Sollner-webb, Barbara.

In: Nucleic Acids Research, Vol. 12, No. 14, 25.07.1984, p. 5707-5717.

Research output: Contribution to journalArticle

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