TY - JOUR
T1 - 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
AU - Lopata, Margaret A.
AU - Cleveland, Don W.
AU - Sollner-webb, Barbara
N1 - Funding Information:
We thank Greg Hilman and Dan Sussnan for motivating as to analyze the level of gene expression after DEAE-dextran and DMSO mediated trsnsfection. Wo also thank Bruce Howard for the pSV2-CAT and pSVO-CAT plasmids and George Khoury for the mouse L cells. This work was supported by NIH grants GM27720 and GM29513 and Basil O'Connor March of Dines Research Grants 5-386 and 5-406.
PY - 1984/7/25
Y1 - 1984/7/25
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/12.14.5707
DO - 10.1093/nar/12.14.5707
M3 - Article
C2 - 6589587
AN - SCOPUS:0021770787
SN - 0305-1048
VL - 12
SP - 5707
EP - 5717
JO - Nucleic acids research
JF - Nucleic acids research
IS - 14
ER -