Activation of the control reporter plasmids pRL-TK and pRL-SV40 by multiple GATA transcription factors can lead to aberrant normalization of transfection efficiency.
BACKGROUND
Members of the GATA transcription factor family have been used in many transfection studies to investigate their roles in the regulation of gene expression. To correct for variations in transfection efficiency, the Renilla luciferase encoding plasmids pRL-TK and pRL-SV40 are commonly used as normalization controls.


RESULTS
We report here that plasmids expressing GATA-4 or GATA-6 transcription factor increased Renilla luciferase gene expression by 2 to 8 fold when co-transfected with pRL-TK or pRL-SV40. This alteration of the control reporter gene activity was shown to cause erroneous normalization of transfection efficiency and thus misinterpretation of results in a transactivation assay. To circumvent the problem, we generated two mutant control plasmids from which putative GATA response elements were deleted. These deletions rendered pRL-SV40 unresponsive to GATA transcription factor stimulation and reduced the response of pRL-TK. A database search also indicates that consensus GATA binding sequences are present in other commercially available Renilla luciferase encoding plasmids; therefore, the latter can potentially be transactivated by GATA transcription factors.


CONCLUSIONS
Taken together, these findings highlight the importance of the selection of an appropriate control reporter plasmid for the normalization of transfection efficiency.
Ho
Clement K M
CK
Center for Research on Reproduction and Women's Health, University of Pennsylvania School of Medicine, 1355 Biomedical Research Building II/III, 421 Curie Blvd, Philadelphia, PA 19104, USA. clement.ho@doctors.org.uk
Strauss
Jerome F
JF
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000378:metabolism; D050991:GATA4 Transcription Factor; D050993:GATA6 Transcription Factor; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D050993:GATA6 Transcription Factor; D014157:Transcription Factors; D008156:Luciferases; D013937:Thymidine Kinase