les_2 - CHAPTER II: REPORTER SYSTEMS AND DETECTION METHODS...

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21 CHAPTER II: REPORTER SYSTEMS AND DETECTION METHODS During their research, a lot of investigators are confronted with the fact that their protein of interest on its own has no enzymatic or biochemical activity or can not be easily visualized or assayed (e.g. due to lack of good antibodies). Direct quantification of gene expression, protein localization is thus indeed not always possible. On the other hand, biotech and pharmaceutical companies often rely on good high throughput assays e.g. to screen for novel drugs. In both cases, reporter genes and systems may provide a solution for these problems. A reporter gene is a gene whose phenotypic expression is easy to monitor. Recombinant DNA constructs are made in which the reporter gene is attached to a promoter region of particular interest and the construct is transfected into a cell or organism. In some cases, like e.g. the reporter gene GFP, the gene expressing your protein of interest is cloned in frame with the reporter and visualized/monitored in this way. Reporter genes are generally used to determine whether the gene of interest has been taken up by or is expressed in the cell, tissue or organism. II.1. WHY USE REPORTER SYSTEMS? The applications for reporter genes are diverse and are widely used to measure the effects of experimental conditions on cellular physiology e.g. -to check transfection processes/efficiency (e.g. β -gal expression in specific cells) -to follow up genetic construction processes (e.g. blue/white screening) - to analyze and detect gene transcription and regulation: promotor/enhancer activity and regulatory events (e.g. effect of inhibitors/activators on promotor activity, to determine essential TF- binding sites within a promotor,…)
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- to analyze localization and time of expression of protein of interest within cells, tissues or even within whole organisms in situ or in vivo (e.g. by PET (positron emission tomography) analysis) -to study signaling pathways (e.g. luciferase reporter assays to check activation of transcription factors) -to detect recombination events (e.g. Cre/Lox) -as readout for protein-protein interaction (e.g. X-gal colouring in Y2H assays) In this chapter we will try to outline the most used reporter systems and recent developments, with their advantages and disadvantages as well as their use in Molecular Biology. II.2. BASIC PRINCIPLES AND REQUIREMENTS OF REPORTER SYSTEMS -reporter gene product detection should be easy, cheap and fast (often used for high- through put assays in biotech companies) -reporter half-life (e.g. dynamics of gene expression versus transfection efficiency) - reporter genes and their activity can be visualized or quantified by 1. luminescence 2. fluorescence 3. radio activity 4. colorimetry/spectrophotometry 5. Abs: ELISA, WB -two types of reporter systems: reporter can be detected without substrate addition (e.g. fluorescent proteins like GFP) or reporter is an enzyme and is detected by substrate
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les_2 - CHAPTER II: REPORTER SYSTEMS AND DETECTION METHODS...

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