A high-throughput mammalian cell-based transient transfection assay.
In eukaryotic organisms gene expression is regulated through a variety of upstream transacting factors (transcription factors) whose primary function appears to be the targeting of coregulatory protein complexes, which interact with basal transcription machinery to define the relative rate of transcription for a specific gene. Understanding the regulatory forces mediating transcription factor activity has been the focus of both academic and industrial research efforts over the past 15 yr, and in this time frame a variety of methodologies have been developed for reconstituting and assaying transcription factor activities in mammalian cell environments. Presented here is a high-throughput version of one of these methodologies that can be readily adapted to the screening of a variety of transcription factors. This technology utilizes co-transfection of mammalian expression and luciferase reporter plasmids to reconstitute transcription events in a mammalian host cell. Included is a detailed protocol for the use of a 96-well plate format, along with a variety of cost-effective measures that can be implemented to facilitate the use of the technology in the average low budget academic laboratory.
Noonan
Daniel J
DJ
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, USA.
Henry
Kenneth
K
Twaroski
Michelle L
ML
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D008163:Luminescent Measurements; D010957:Plasmids / Q000235:genetics; D012371:Robotics / Q000295:instrumentation; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D014157:Transcription Factors; D008156:Luciferases