Quantitation of transient gene expression after electroporation.
The combination of a photometric reporter-gene assay, with transfection by electroporation, is potentially a rapid and sensitive tool for the study of genetic regulatory elements in many types of cells. We have found that the sensitivity, accuracy, and reproducibility of the technique is greatly improved by the inclusion of appropriately chosen carrier DNA as the primary DNA species present during electroporation. By using high levels of carrier, the activities of constructs of differing sizes can be quantitatively compared, active constructs can be assayed with sub-microgram amounts of plasmid, and the activities of the constructs are linear over a wide concentration of DNA. In addition, the activity of miniprep DNA can be screened without purification on CsCl gradients giving activities equal to CsCl-purified DNA. This is extremely useful when doing preliminary screening of large numbers of constructs for promoter or enhancer activities. We report the results of testing various types of DNA as carrier, and the parameters for optimizing its use.
Showe
M K
MK
Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104.
Williams
D L
DL
Showe
L C
LC
D003001:Cloning, Molecular / Q000379:methods*; D004558:Electric Stimulation; D004586:Electrophoresis; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D008156:Luciferases; D001616:beta-Galactosidase