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 / 中华肝脏病杂志
Chinese Journal of Hepatology
;
(12): 747-751, 2014.
Article
in Chinese
| WPRIM
| ID: wpr-337106
ABSTRACT
<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Base Sequence
/
Transfection
/
Transcriptional Activation
/
Promoter Regions, Genetic
/
Genes, Reporter
/
Collagen Type I
/
Drug Evaluation, Preclinical
/
Drug Therapy
/
Transforming Growth Factor beta1
/
Genetic Vectors
Type of study:
Diagnostic study
/
Prognostic study
/
Screening study
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Hepatology
Year:
2014
Type:
Article
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