Les2_complete_ppt_blackwhite

Les2_complete_ppt_blackwhite - Chapter II: Reporter systems...

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Chapter II: Reporter systems and detection methods
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Outline 1. What? Why? 2. Basic principles 3. Common reporter systems: 1. What, detection 2. Applications, recent developments 3. Advantages/disadvantages
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1. What? A reporter gene = gene whose phenotypic expression is easy to monitor/visualize
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Why were reporter systems developed? • Protein of interest: – No biochemical activity – No enzymatic activity – Hard to visualize – Lack of good antibodies – …. . • Companies: HT assays
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To control. .. To control. .. – genetic construction process genetic construction process (blue/white screen) (blue/white screen) – transfection transfection processes and efficiency processes and efficiency To detect gene expression and regulation To detect gene expression and regulation – Promotor Promotor /enhancer activity /enhancer activity – Regulatory events Regulatory events ( e.g. effect of inhibitors on promotor activity, to determine essential TF-binding sites within a promotor,…) – Recombination events ( Recombination events ( Cre Cre /Lox /Lox ) To visualize cellular processes To visualize cellular processes (( (( trans)location trans)location , PPI, signaling, , PPI, signaling, …) Why use reporter systems? Hey! I’ve been produced by this cell. ..
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2. Basic principles/requirements • Cheap, easy and fast (HT!!) • Reporter half-life • Reporter gene (activity) quantification and visualization: lum, fl, RA, color, WB, ELISA
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• Reporter: – Reporter could be detected Reporter could be detected as such as such without without substrate substrate addition addition (e.g. fluorescent proteins) (e.g. fluorescent proteins) – Reporter = Reporter = enzyme enzyme : detected by substrate conversion : detected by substrate conversion (e.g. (e.g. β -Gal) Gal) • Easy substrate delivery Easy substrate delivery • No intrinsic substrates should exist No intrinsic substrates should exist • Substrates should not be converted by intrinsic Substrates should not be converted by intrinsic enzyme activity enzyme activity Reporter from other species Reporter from other species • Secreted Secreted 2. Basic principles/requirements
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Reporter system build-up: gene,P or TF Gene of Interest P P Gene of Interest Reporter P STOPP Reporter Cre Cre TF P Reporter P Reporter
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3. Reporter systems 1. Chloramphenicol Ac transferase (CAT) 2. β -galactosidase ( β -gal) 3. Luciferase (luc) 4. Secreted alkaline phosphatase (SEAP) 5. (Green) Fluorescent proteins ((G)FP)
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Task • Info and figures from articles or brochures from companies selling the reporter system
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This note was uploaded on 05/28/2010 for the course WE BIBI000000 taught by Professor Johangrooten during the Spring '10 term at Ghent University.

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Les2_complete_ppt_blackwhite - Chapter II: Reporter systems...

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