The use of a second reporter plasmid as an internal standard to normalize luciferase activity in transient transfection experiments may lead to a systematic error.
beta-galactosidase reporter plasmids containing different viral or minimal promoters are commonly used to correct variable transfection efficiencies in transient transfection experiments. The transcriptional activity of these promoters is thought to be stable under most circumstances. To determine if expression of beta-galactosidase from the commonly used beta-galactosidase plasmids remains stable upon stimulation of the cells with agonists we performed transient transfection experiments. CHO cells stably expressing the rat AT(1A) receptor were transfected with RSVbeta- or CMVbeta- or pTKbeta plasmids alone or together with a reporter construct in which luciferase transcription is driven by the c-fos promoter. Luciferase and/or beta-galactosidase activity was measured from the lysate of cells treated with angiotensin II or serum. We found that agonists increased the transcriptional activity of the different beta-galactosidase plasmids. The effect of angiotensin II and serum was different on the different promoters. Finally, cotransfection of other plasmids also modulated beta-galactosidase activity. These agonist induced variations of beta-galactosidase activity may influence the analysis and interpretation of the results in a systematic manner. Consequently we conclude that the use of a second reporter system to control for transfection efficiency in certain types of experiments may lead to a systematic error and is questionable as a general procedure.
Huszár
T
T
Department of Pathophysiology, Faculty of Medicine and Nephrology Training and Research Center, Semmelweis University, 18th Floor, Nagyvárad tér 4, H-1089, Budapest, Hungary.
Mucsi
I
I
Terebessy
T
T
Masszi
A
A
Adamkó
S
S
Jeney
C
C
Rosivall
L
L
D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols; D002130:Calcium Phosphates / Q000737:chemistry; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003520:Cyclophosphamide; D004317:Doxorubicin; D015870:Gene Expression; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D016762:Genes, fos; D008156:Luciferases / Q000235:genetics*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000378:metabolism; D012015:Reference Standards; D015398:Signal Transduction; D014162:Transfection; D014750:Vincristine; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics*
D002130:Calcium Phosphates; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D014750:Vincristine; D004317:Doxorubicin; D003520:Cyclophosphamide; D008156:Luciferases; D001616:beta-Galactosidase