Nurr1 affects pRL-TK but not phRG-B internal control plasmid in genetic reporter system.
In transcription assays, Renilla luciferase-expressing plasmids (more specifically pRL-TK) are commonly used as an internal control of transfection efficiency. Normalization of the experimental reporter gene transcription to the internal control reporter gene transcription minimizes variability of obtained results caused by differences in transfection efficiency between different samples of transfected cells. It is obvious that co-transfection with other plasmids or applied treatments should not affect the activity of the control reporter. Here we report that expression of the control Renilla luciferase encoded by pRL-TK plasmid was enhanced by co-transfection with vectors expressing orphan nuclear receptors Nur77 family (Nur77, Nurr1, Nor-1), leading to misinterpretation of the assay results. Further, we show that for Nurr1, phRG-B (a promoterless reporter plasmid containing synthetic Renilla luciferase gene) is a better control reporter vector than HSV-TK containing vectors. Finally, we noted the lack of effect of Nurr1 protein on the Fas Ligand promoter-driven transcription.
Matuszyk
Janusz
J
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigl 12, 53-114 Wroclaw, Poland. matuszyk@iitd.pan.wroc.pl
Ziolo
Ewa
E
Cebrat
Malgorzata
M
Kochel
Izabela
I
Strzadala
Leon
L
D000818:Animals; D003063:Cnidaria / Q000201:enzymology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D016716:PC12 Cells; D010957:Plasmids; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D012015:Reference Standards; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods* / Q000592:standards
D004268:DNA-Binding Proteins; C539820:Nr4a1 protein, rat; C539884:Nr4a2 protein, rat; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D014157:Transcription Factors; D008156:Luciferases