title: [Construction and validation of a dual-luciferase reporter gene system for screening and evaluating anti-liver fibrosis drugs that inhibit transcription of the gene encoding collagen I, chain a1].
abstract: OBJECTIVE
  To construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain at1 (COL1A1).
  
  
  METHODS
  The full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter.All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing.Either the pcDNA3.1-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter gene system.This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1.Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system.
  
  
  RESULTS
  The two TGFbeta1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed.The results of a dual-reporter gene assay showed that TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 co-expression the activity was below 2-fold (t =3.396, P =0.0274).The transcriptionactivated dual-reporter gene system was successfully established.The model drug, dexamethasone, effectively inhibited the activity of the COL 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035).
  
  
  CONCLUSIONS
  The dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.
authors.affiliation: Department of Liver Surgery, Renji Hospital School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Dai
authors.forename: Hong
authors.initials: H
authors.lastname: Yu
authors.forename: Luyan
authors.initials: L
authors.lastname: Wang
authors.forename: Shihui
authors.initials: S
authors.lastname: Sun
authors.forename: Junfeng
authors.initials: J
authors.lastname: Li
authors.forename: Yusen
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D001483:Base Sequence; D024042:Collagen Type I / Q000235:genetics*; D000090602:Collagen Type I, alpha 1 Chain; D004353:Drug Evaluation, Preclinical; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008103:Liver Cirrhosis / Q000188:drug therapy*; D008156:Luciferases; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D053773:Transforming Growth Factor beta1
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D053773:Transforming Growth Factor beta1; D008156:Luciferases