Enhancing quantification of mammalian cell transfections with chloramphenicol acetyltransferase reporter plasmids.
We modified existing techniques to optimize conditions for obtaining quantitative, highly replicable, and sensitive transfections. The processes described may serve as a model for investigators initiating transfection procedures who wish to obtain definitive and quantitative results quickly and efficiently. In our example, we compared specific gene expressions of plasmids with the chloramphenicol acetyltransferase (CAT) reporter. Techniques included measuring CAT activity in transfected mammalian cells, selecting a procedure for extracting plasmids from bacterial cells, evaluating the timing of the transfection, choosing a transfection reagent and the reagent: plasmid DNA ratio, and determining procedures for the extraction of cells.
Fernandez
P M
PM
Department of Pharmacology, The George Washington University Medical Center, 2300 Eye St., NW, Washington, DC 20037, USA.
Mandel
H G
HG
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011933:Reagent Kits, Diagnostic; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D011933:Reagent Kits, Diagnostic; D015500:Chloramphenicol O-Acetyltransferase