Abstract：
ReViTA (Reverse in VitroTranscription Assay) is a novel in vitro transcription-based method to study gene expression under the regulation of specific transcription factors. The ReViTA system uses a plasmid with a control sequence, the promoter region of the studied gene, the transcription factor of interest, and an RNA polymerase saturated with σ70. The main objective of this study was to evaluate the method; thus, as a proof of concept, two different transcription factors were used, a transcriptional inducer, AlgR, and a repressor, LexA, from Pseudomonas aeruginosa. After the promoters were incubated with the transcription factors, the plasmid was transcribed into RNA and reverse transcribed to cDNA. Gene expression was measured using qRTPCR. Using the ReViTA plasmid, transcription induction of 55% was observed when AlgR protein was added and a 27% transcription reduction with the repressor LexA, compared with the samples without transcription factors. The results demonstrated the correct functioning of ReViTA as a novel method to study transcription factors and gene expression. Thus, ReViTA could be a rapid and accessible in vitro method to evaluate genes and regulators of various species.
摘要：
ReViTA(ReverseinVitroTranscriptionAssay)是一种新的基于体外转录的方法，用于研究特定转录因子调控下的基因表达。ReViTA系统使用具有控制序列的质粒，所研究基因的启动子区域，感兴趣的转录因子，和用σ70饱和的RNA聚合酶。本研究的主要目的是评估该方法；因此，作为概念的证明，使用了两种不同的转录因子，转录诱导剂，AlgR,和抑制物，LexA,来自铜绿假单胞菌.启动子与转录因子孵育后，将质粒转录为RNA并逆转录为cDNA。使用qRTPCR测量基因表达。使用ReViTA质粒，当添加AlgR蛋白时观察到55％的转录诱导和27％的转录减少与抑制因子LexA，与没有转录因子的样品相比。结果证明了ReViTA作为研究转录因子和基因表达的新方法的正确功能。因此，ReViTA可能是一种快速且易于使用的体外方法，可用于评估各种物种的基因和调节因子。
