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Tetracycline-based Control System to Construct Regulated Eukaryotic Expression System

Author: ZhangXiaoNan
Tutor: ChenShan；ChenHuiPeng
School: Northeast Normal University
Course: Microorganism
Keywords: tTS / tet-on Strict regulation of Cell model Stable expression
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 90
Quote: 0
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Abstract


Regulation of gene expression is a molecular biology research is an important issue. Especially at the transcriptional level, and its regulation is a very complex process. Currently there are some problems to study gene expression and regulation caused difficulties, such as the continuous expression of some genes is close to cytotoxic levels can cause cell death, toxicity, lack of precise study of gene function control systems, as well as the goal to produce a toxic protein The engineered cells difficult issues such as the construction. Therefore construct a rigorous, efficient and regulated inducible gene expression system, can be artificially controlled exogenous gene expression levels, is conducive to simulate the gene under physiological and pathological conditions, such as different characteristics and mechanism of action. Using this inducible regulatory system can be used for quantitative regulation of gene expression and gene function analysis for toxicity studies. All current regulation of eukaryotic gene expression system tetracycline regulatory system (tet systerm) is considered to be the most ideal eukaryotic gene regulation system. Wherein the tetracycline-regulated system tTS / tet-on control systems most widely used, the system is composed of: two regulatory protein (tTS, rtTA) expression cassette, and a tetracycline response (TRE), the system inducer tetracycline (Tc ) or its derivative doxycycline (Dox). Joint use of two kinds of regulatory proteins not only will not affect the system's ability to induce its basal expression levels can be greatly reduced, is already widely used to build various cellular models to study the function of the gene being. In this study, a new tTS / tet-on regulation of the expression vector, selected CHO-K1 host cell, constructed and screened stable tTS / tet-on cell lines. Through Real-time PCR and dual luciferase reporter assay system for screening out tTS / tet-on regulation of cell lines expressing the regulation of performance were measured and compared. Real-time PCR results and luciferase measurement data show that screened tTS / tet-on regulation of cell lines expressing more rigorous and efficient, and achieved good results. On this basis, the use of screened tTS / tet-on regulation of expression of cell lines for virus 2A protease gene expression and regulation studies to prove rigorous type tTS / tet-on regulation of expression of cell lines can be achieved on the regulation of toxic protein expression . The study successfully constructed and screened two rigorous type tTS / tet-on regulation of cell lines of its activity and low basal expression of the ability to induce a strong interest for the purpose of exogenous genes in eukaryotic cells provided in the regulation of expression a technology platform.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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