Name: reporter genes in cells and plasmids
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 11 s
Channel: JoVE

K12 Standards

Tennessee Academic Standards

High School

Biology

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Reporter Genes

Forscher verwenden Reportergene, um zu bestimmen, wann und wo ein Gen von Interesse exprimiert wird. Ein Reportergen kodiert für ein Protein, das verfolgt werden kann, z. B. ein Protein mit einer bekannten enzymatischen Aktivität oder ein Protein, das fluoreszierend ist.
Die Genexpression wird durch cis-regulatorische Sequenzen gesteuert, die sich stromaufwärts oder stromabwärts der kodierenden Region eines Gens in der DNA befinden.
Das Muster und der Zeitpunkt der Genexpression können bestimmt werden, indem rekombinante DNA mit einem Reportergen unter der Kontrolle einer cis-regulatorischen Sequenz von Interesse erzeugt und in Zellen oder einen Organismus eingeführt wird.
Dieses rekombinante Gen wird in verschiedene Zelltypen eingeführt, und sowohl das interessierende Gen als auch das Reportergen dürfen exprimieren.
Da das interessierende Gen und das Reportergen die gleiche cis-regulatorische Sequenz haben, werden sie in denselben Zellen und zur gleichen Zeit exprimiert. Durch die Überwachung der Expression des Reportergens kann ein Wissenschaftler also verfolgen, wo das interessierende Gen exprimiert wird.
Eines der bekannten Reportergene ist eines, das für ein Protein namens grün fluoreszierendes Protein - oder GFP - kodiert.
GFP wurde in der Qualle Aequorea victoria entdeckt. Das Protein erzeugt unter ultraviolettem Licht eine grüne Fluoreszenz, die es den Forschern ermöglicht, seine Position innerhalb einer Zelle zu verfolgen.
Um beispielsweise die Expression von β-Tubulin in C. elegans zu untersuchen, wurde die kodierende Sequenz des β-Tubulin-Gens durch das gfp-Gen ersetzt.
Die Expression des gfp-Gens steht nun unter der Kontrolle des Promotors für das β-Tubulin-Gen.
Diese rekombinante DNA wurde durch eine Mikroinjektion in den Wurm eingeführt, und das Gen wurde ähnlich wie β-Tubulin exprimiert.
Die Lokalisation der GFP wurde mittels Fluoreszenzmikroskopie überwacht. Da die GFP-Fluoreszenz in den Berührungsrezeptor-Neuronen gezeigt wurde, ist dies wahrscheinlich eine Stelle, an der β-Tubulin exprimiert wird.

<ol>
	<li>Soboleski, Mark R., Jason Oaks und William P. Halford. "Grün fluoreszierendes Protein ist ein quantitativer Reporter der Genexpression in einzelnen eukaryotischen Zellen." The FASEB Journal 19, Nr. 3 (2005): 1-20.</li>
	<li>Brogan, John, Fang Li, Wenrong Li, Zhimin He, Qian Huang und Chuan-Yuan Li. "Bildgebende molekulare Signalwege: Reportergene." Strahlenforschung 177, Nr. 4 (2012): 508-513.</li>
	<li>Serganova, Inna, Vladimir Ponomarev und Ronald Blasberg. "Menschliche Reportergene: potenzieller Einsatz in klinischen Studien." Nuklearmedizin und Biologie 34, Nr. 7 (2007): 791-807.</li>
</ol>

reporter-gene

Reporter-Gene sind eine Art von Protein-kodierenden Genen, die oft an ein Gen von Interesse angehängt werden. Einmal in eine Zielzelle eingebracht, produzieren Reporter-Gene in der Regel visuell identifizierbare Merkmale wie Fluoreszenz und Lumineszenz, wenn sie zusammen mit dem Gen von Interesse exprimiert werden. Reporter-Gene „berichten“ also über die An- oder Abwesenheit von Genen von Interesse in einem Organismus, bestimmen das Genexpressionsmuster oder verfolgen den physischen Standort eines DNA-Segments oder Proteins in der Zelle.

Häufig verwendete Reporter-Gene sind GFP (Gen für grünes fluoreszierendes Protein), lacZ (Gen für β-Galaktosidase), RFP (Gen für rotes fluoreszierendes Protein) und Luc (Gen für Luziferase). Die Zellen, die GFP exprimieren, leuchten grün, wenn sie mit Wellenlängen von Blau bis Ultraviolett angeregt werden, während diejenigen, die RFP exprimieren, rot leuchten, wenn sie mit Wellenlängen von 488 nm oder 532 nm angeregt werden. Die Zellen, die das Luc-Gen exprimieren, produzieren Luziferase-Enzyme, die eine Reaktion mit Luziferin katalysieren, um Licht zu erzeugen. Das lacZ-Gen ist das am häufigsten verwendete Reporter-Gen in E.coli. Es produziert das Enzym β-Galaktosidase, das dazu führt, dass die Bakterien blau erscheinen, wenn sie auf einem Medium mit X-Gal-Substrat wachsen.

Reporter-Gene zur Untersuchung der Promotoraktivität

Neben der Verwendung von Reporter-Genen zur Untersuchung der Genexpression und -lokalisierung können sie auch dazu dienen, die Stärke von Promotoren zu studieren. Dies geschieht, indem ein Reporter-Gen stromabwärts des Promotors in ein Plasmid eingefügt und dann in die Säugetierzelle eingeführt wird. Die Expression des Reporter-Gens überwacht die Aktivität des Promotors. Wenn die Expression des Reporter-Gens hoch ist, wird der Promotor als starker Promotor bezeichnet; er gilt als schwach, wenn die Reporter-Gen-Expression gering ist.

Ein Reporter-Gen, das stromabwärts des Promotors in einem Plasmid platziert wird, hilft auch bei der Messung der Transfektionseffizienz. Das rekombinante Plasmid wird in eine Säugetierzelle transfiziert, und seine Expression zeigt, dass die Zelle erfolgreich transfiziert wurde. Die Menge an Protein, die durch das rekombinante Plasmid produziert wird, zeigt seine Transfektionseffizienz.

Reporter-Gene

Reportergene sind eine Art von proteinkodierenden Genen, die oft mit einem Gen von Interesse verknüpft sind. Einmal in einer Zielzelle angekommen, ...

Education

Lehre

JoVE Core

Molecular Biology

Molekularbiologie

Unit 2

Einheit 2

Analyzing Gene Expression and Function

Analyse von Genexpression und -funktion

KEY TERMS AND CONCEPTS

KERNBEGRIFFE UND KONZEPTE

reporter genes in cells and plasmids

Reporter genes are protein-coding genes used to track gene activity in cells. They are often tagged to a gene of interest so scientists can see whether that gene is present or active. When the tagged genes are expressed together, reporter genes can produce visible signals such as fluorescence or luminescence.

These signals help show the presence or absence of a gene of interest in an organism. They can also reveal the pattern of gene expression or the location of a DNA segment or protein inside a cell. Common reporter genes include GFP, lacZ, RFP, and Luc.

GFP stands for green fluorescent protein gene. Cells that express GFP glow green when exposed to blue to ultraviolet light. RFP stands for red fluorescent protein gene, and cells that express it glow red when excited by 488 nm or 532 nm light. The Luc gene produces luciferase enzymes, which react with luciferin to make light. The lacZ gene is the most common reporter gene used in E. coli. It produces β-galactosidase, which makes bacteria appear blue in media that contains X-gal substrate.

Reporter genes are also useful for studying promoter activity. A promoter is a DNA region that controls when a gene is turned on. Scientists place a reporter gene downstream of the promoter in a plasmid and then introduce the plasmid into a mammalian cell. The amount of reporter gene expression shows how active the promoter is. High expression suggests a strong promoter, while low expression suggests a weak promoter. Reporter genes can also help measure transfection efficiency, which shows whether the recombinant plasmid entered the mammalian cell successfully and how much protein it produced.

Reporter Genes in Cells and Plasmids

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reporter genes

Reporter genes are protein-coding genes used to track gene activity in cells. They are often tagged to a gene of interest so scientists can see whether that gene is present or active. When the tagged genes are expressed together, reporter genes can produce visible signals such as fluorescence or luminescence. These signals help show the presence or absence of a gene of interest in an organism. They can also reveal the pattern of gene expression or the location of a DNA segment or protein...

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transgenic plants

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key terms and concepts

Learn how reporter genes like GFP, lacZ, RFP, and Luc reveal gene expression, promoter strength, and transfection efficiency in cells and plasmids.

Reporter Genes in Cells and Plasmids | JoVE K12

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Reporter genes are a type of&#160;protein-coding gene&#160;that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually ...

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BIO1.ETS2.3

Reporter Genes in Cells and Plasmids

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