Factors influencing transient expression in cytotoxic T cells following DEAE dextran-mediated gene transfer.
A number of transfection protocols have been tested for the introduction of exogenous DNA into cytotoxic T cells. These included electroporation, lipofection, calcium phosphate coprecipitation, polybrene-assisted gene transfer, and DEAE dextran-mediated transfer. Only the latter gave significant and reproducible transfection efficiencies coupled with low toxicity. The DEAE dextran protocol was optimized for the transfection of a transcription reporter construct pRSVcat into a cloned cytotoxic cell line. Among the parameters investigated were cell density, amount of input DNA, concentration of DEAE dextran, DNA adsorption time, temperature, use of permeabilization and expression facilitators, and recovery time. The optimized protocol was then used to demonstrate the presence of cis-acting regulatory regions in the 5'-flanking sequences of two cytotoxic cell-specific serine protease genes and, in addition, was shown to be applicable to other cloned T-cell lines.
Frégeau
C J
CJ
Department of Biochemistry, University of Alberta Edmonton, Canada.
Bleackley
R C
RC
D000327:Adsorption; D000818:Animals; D002130:Calcium Phosphates / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability; D011232:Chemical Precipitation; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003637:DEAE-Dextran; D004247:DNA / Q000378:metabolism; D004553:Electric Conductivity; D015870:Gene Expression; D006583:Hexadimethrine Bromide / Q000494:pharmacology; D007700:Kinetics; D007958:Leukocyte Count; D051379:Mice; D008807:Mice, Inbred BALB C; D008808:Mice, Inbred CBA; D011933:Reagent Kits, Diagnostic; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection / Q000187:drug effects*
D002130:Calcium Phosphates; D011933:Reagent Kits, Diagnostic; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D006583:Hexadimethrine Bromide; C485838:calcium phosphate, monobasic, anhydrous; D004247:DNA; D003637:DEAE-Dextran; C020243:calcium phosphate; D015500:Chloramphenicol O-Acetyltransferase; C485829:calcium phosphate, dibasic, anhydrous