Stealth sequences in reporter gene vectors confound studies of T3-regulated negative gene expression.
Stealth sequences in reporter gene vectors confound studies of T3-regulated negative gene expression.
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报告基因载体中的隐形序列混淆了 T3 调节负基因表达的研究。
DOI：
10.1089/thy.2008.0138
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Brent,GregoryA
中科院分区：
文献类型：
--
作者：
Liu,Yan-Yun;Brent,GregoryA
Firefly luciferase is a commonly used reporter gene that allows for rapid and precise quantitation of gene expression. It is utilized in a wide range of vectors to evaluate many aspects of transcription regulation. The native luciferase gene (luc), however, contains DNA sequences that are recognized binding sites for a large number of transcription factors (Table 1).In this issue of Thyroid, Chan et al. report that the pBi-L vector (Clontech, Mountain View, CA) is negatively regulated by triiodothyronine (T3)(1). In a series of transfection studies, utilizing several cell lines, they demonstrate that negative regulation is conferred by sequences in the luciferase gene and that expression of thyroid hormone receptor (TR) is required. The pBi-L vectors are tetracycline-regulated reporters. The luciferase gene sequence in pBi-L is 99% identical to that in the pGL-2 reporter series (Promega, Madison, WI), which carries a minimally modified native luc. The expression of the luciferase gene in the pGL-2 promoter vector was previously reported to be inhibited by T3 treatment in CV1 and JEG3 cells (2, 3), thought to be mediated by a negative thyroid hormone response element (nTRE) in the luciferase gene. Luciferase gene expression in Caco-2 cells is inhibited by 1, 25-dihydroxycholecalciferol when using the pGL-2 promoter vector, but not when using the pGL-3 promoter vector (4). In the pGL-3 reporter series, luciferase gene (lucþ) is modified from native luc in order to reduce anomalous expression and enhance Lucþ expression. Even with this modification, however, the pGL-3 series still carries numerous binding sites for transcription factors (Table 1). Examples of these include elements for CREB, CAR, SRF, PAR, RXR, HNF, Oct, CAR, CHOP, and other factors (Table 1). We have reported that lucþ expression is induced by alltrans retinoic acid up to fourfold in MCF-7 cells (5). The further modified version of the luciferase gene (luc2) used in the pGL-4 reporter vector series dramatically reduces the number of transcription factor binding sites, but binding sites for a few common transcription factors remain (Table 1). Despite several reports of T3-dependent repression mediated by sequences in the luciferase gene, the nTRE sequence in the luciferase gene has not been identified. The sequence configurations that confer positive regulation have been well defined (6), but the mechanism of negative regulation by T3 is more complex. The ability of an element to confer negative regulation is influenced by both sequence and position (7). In models involving TR directly binding to an nTRE for T3-dependent repression, a variety of sequences
