Tetracycline-based Control System To Construct Regulated Eukaryotic Expression System

Gene expression and regulation is an important area in Molecular Biology, particularly in the level of transcription, its regulation is a very complex process.At present some problems caused difficulties for gene expression and regulation.Such as the continued expression of certain genes are toxic to cells and cause cell death, the lack of accurate system to study virulence gene function,and the production of toxic proteins targeted cell line engineering construction,etc.To construct a tight, effective and inducible gene regulatory expression system for human-controlled gene expression levels,it conducive to simulate the gene in physiological and pathological roles of different state characteristics and mechanisms.Using this inducible system can be used for quantitative regulation of gene expression regulation and virulence gene function analysis and so on.Tetracycline control system (tet systerm) is regarded as the most ideal eukaryotic gene expression system in all eukaryotic gene regulatory exression system.Nowadays the most frequently and widely used eukaryotic gene regulatory expression system is the tTS/tet-on system of tet systems. This regulatory system consisted of: two regulatory proteins(tTS,rtTA), and a response element(TRE),inducer of the system is the tetracycline (Tc) or its derivatives doxycycline (Dox).The joint use of two kinds of regulatory proteins are not only will affect the ability to induce,but also the basis of their expression levels can be greatly reduced.Now it has been widely used in building a variety of cell models to study gene function.In this study, according to a new building completed tTS/tet-on regulation of expression vector,choose CHO-K1 as the host cell to build and filt stable tTS/tet-on cell line.By Real-time PCR and firefly luciferase as the reporter to have the constructed and filted tTS/tet-on cell lines been compared in the field of regulation capabilities. The data of Real-time PCR anf firefly luciferase indicate that screened tTS/tet-on cell lines with lower expression level and more effective,achieved a good results.In this study,we also try to regulate the synthesis of toxic protein 2Apro by screened this new type of tTS/tet-on gene regulatory expression cells by preliminary discussion the results,the tight type tTS/tet-on cell lines can be achieved on the expression and regulation of toxic protein.This study has successfully constructed and screened two tTS/tet-on gene regulatory expression cells.Its lower expression level and high inductive ability could provides a technology platform for the tightly regulatory expression of.exogenous protein in eukaryotic cells.