Activation of the control reporter plasmids pRL-TK and pRL-SV40 by multiple GATA transcription factors can lead to aberrant normalization of transfection efficiency

被引:42
作者
Ho, CKM [1 ]
Strauss, JF [1 ]
机构
[1] Univ Penn, Sch Med, Ctr Res Prod & Womens Hlth, Philadelphia, PA 19104 USA
关键词
D O I
10.1186/1472-6750-4-10
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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. Conclusion: Taken together, these findings highlight the importance of the selection of an appropriate control reporter plasmid for the normalization of transfection efficiency.
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页数:5
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共 9 条
[1]   The human and mouse GATA-6 genes utilize two promoters and two initiation codons [J].
Brewer, A ;
Gove, C ;
Davies, A ;
McNulty, C ;
Barrow, D ;
Koutsourakis, M ;
Farzaneh, F ;
Pizzey, J ;
Bomford, A ;
Patient, R .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1999, 274 (53) :38004-38016
[2]   Nurr1 affects pRL-TK but not phRG-B internal control plasmid in genetic reporter system [J].
Matuszyk, J ;
Ziolo, E ;
Cebrat, M ;
Kochel, I ;
Strzadala, L .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2002, 294 (05) :1036-1039
[3]   The zinc finger-containing transcription factors GATA-4,-5, and-6 - Ubiquitously expressed regulators of tissue-specific gene expression [J].
Molkentin, JD .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (50) :38949-38952
[4]   GATA-6: A zinc finger transcription factor that is expressed in multiple cell lineages derived from lateral mesoderm [J].
Morrisey, EE ;
Ip, HS ;
Lu, MM ;
Parmacek, MS .
DEVELOPMENTAL BIOLOGY, 1996, 177 (01) :309-322
[5]  
Orkin SH, 1998, INT J DEV BIOL, V42, P927
[6]   pRL-TK induction can cause misinterpretation of gene promoter activity [J].
Osborne, SA ;
Tonissen, KF .
BIOTECHNIQUES, 2002, 33 (06) :1240-1242
[7]   Normalization of luciferase reporter assays under conditions that alter internal controls [J].
Sims, RJ ;
Liss, AS ;
Gottlieb, PD .
BIOTECHNIQUES, 2003, 34 (05) :938-940
[8]   GATA factors differentially activate multiple gonadal promoters through conserved GATA regulatory elements [J].
Tremblay, JJ ;
Viger, RS .
ENDOCRINOLOGY, 2001, 142 (03) :977-986
[9]   The molecular phenotype of polycystic ovary syndrome (PCOS) theca cells and new candidate PCOS genes defined by microarray analysis [J].
Wood, JR ;
Nelson, VL ;
Ho, C ;
Jansen, E ;
Wang, CY ;
Urbanek, M ;
McAllister, JM ;
Mosselman, S ;
Strauss, JF .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (29) :26380-26390