Gene transfer by electroporation, lipofection, and DEAE-dextran transfection: compatibility with cell-sorting by flow cytometry.
The aim of this work was to define a transfection procedure that is compatible with the sorting and propagation of cells that transiently express a heterologous gene. Three requirements were established for the procedure and were met with COS monkey kidney cells that express a recombinant glutathione S-transferase (GST) gene. The transfection procedure used had to generate (i) populations in which at least 10% of the cells expressed recombinant GST, (ii) cellular morphological homogeneity throughout the population, and (iii) viable cells with at least a 5% colony-forming ability. Of the transfection techniques tested, only electroporation satisfied all three requirements. Usually 20-22% of the cells that survived electroporation expressed recombinant GST 3 days after electroporation as measured by flow cytometry, and 25% of the cells that survived electroporation formed colonies in cloning assays. Transfection with DEAE-dextran and chloroquine did enable 40% of the surviving cells to express GST, but only 0.01% of the cells that survived transfection formed colonies in cloning assays. Finally, with lipofection, only 1% of the surviving cells expressed recombinant GST, although 25-40% of the cells that survived transfection formed colonies. These studies define the merits and limitations of transfection techniques relative to the analysis and sorting of transfected cells by flow cytometry.
Puchalski
R B
RB
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Fahl
W E
WE
D000818:Animals; D002460:Cell Line; D002469:Cell Separation; D003637:DEAE-Dextran / Q000494:pharmacology; D004560:Electricity; D005434:Flow Cytometry; D005821:Genetic Techniques; D010714:Phosphatidylethanolamines; D014162:Transfection
D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine; D003637:DEAE-Dextran