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

Les chercheurs utilisent des gènes rapporteurs pour déterminer quand et où un gène d’intérêt est exprimé. Un gène rapporteur code pour une protéine qui peut être suivie, telle qu’une protéine ayant une activité enzymatique connue ou une protéine fluorescente.
L’expression des gènes est contrôlée par des séquences cis-régulatrices situées en amont ou en aval de la région codante d’un gène dans l’ADN.
Le modèle et le moment de l’expression génique peuvent être déterminés en créant de l’ADN recombinant avec un gène rapporteur sous le contrôle d’une séquence cis-régulatrice d’intérêt et en l’introduisant dans des cellules ou un organisme.
Ce gène recombinant est introduit dans différents types de cellules, et le gène d’intérêt et le gène rapporteur sont autorisés à s’exprimer.
Le gène d’intérêt et le gène rapporteur ayant la même séquence cis-régulatrice, ils sont exprimés dans les mêmes cellules et en même temps. Ainsi, en surveillant l’expression du gène rapporteur, un scientifique peut suivre l’endroit où le gène d’intérêt est exprimé.
L’un des gènes rapporteurs bien connus est celui qui code pour une protéine appelée protéine fluorescente verte - ou GFP.
La GFP a été découverte chez la méduse, Aequorea victoria. La protéine produit une fluorescence verte sous la lumière ultraviolette, ce qui permet aux chercheurs de suivre son emplacement dans une cellule.
Par exemple, pour étudier l’expression de la β-tubuline chez C. elegans, la séquence codante du gène de la β-tubuline a été remplacée par le gène gfp.
L’expression du gène gfp est maintenant sous le contrôle du promoteur du gène de la β-tubuline.
Cet ADN recombinant a été introduit dans le ver à l’aide d’une micro-injection, et le gène a été exprimé, de la même manière que la β-tubuline.
L’emplacement de la GFP a été surveillé à l’aide de la microscopie à fluorescence. Comme la fluorescence de la GFP a été démontrée dans les neurones des récepteurs tactiles, il s’agit probablement d’un site où la β-tubuline est exprimée.

<ol>
	<li>Soboleski, Mark R., Jason Oaks et William P. Halford. « La protéine fluorescente verte est un rapporteur quantitatif de l’expression génique dans les cellules eucaryotes individuelles. » La revue FASEB 19, n° 3 (2005) : 1-20.</li>
	<li>Brogan, John, Fang Li, Wenrong Li, Zhimin He, Qian Huang et Chuan-Yuan Li. « Imagerie des voies moléculaires : gènes rapporteurs. » Recherche sur les radiations 177, n° 4 (2012) : 508-513.</li>
	<li>Serganova, Inna, Vladimir Ponomarev et Ronald Blasberg. « Gènes rapporteurs humains : utilisation potentielle dans les études cliniques. » Médecine nucléaire et biologie 34, n° 7 (2007) : 791-807.</li>
</ol>

gènes rapporteurs

Les gènes rapporteurs sont un type de gène codant pour une protéine qui est souvent associé à un gène d'intérêt. Une fois à l’intérieur d’une cellule cible, les gènes rapporteurs produisent généralement des caractéristiques visuellement identifiables telles que la fluorescence et la luminescence lorsqu’ils sont exprimés avec le gène d’intérêt. Ainsi, les gènes rapporteurs « signalent » la présence ou l’absence de gènes d’intérêt dans un organisme, déterminent le modèle d’expression génique ou suivent l’emplacement physique d’un segment d’ADN ou d’une protéine dans la cellule.

Les gènes rapporteurs couramment utilisés sont GFP (gène de la protéine fluorescente verte), lacZ (gène de la β-galactosidase), RFP (gène de la protéine fluorescente rouge) et Luc (gène de la luciférase). Les cellules exprimant la GFP brillent en vert lorsqu'elles sont excitées par des longueurs d'onde bleues jusqu'à la plage ultraviolette, tandis que celles exprimant la RFP brillent en rouge lorsqu'elles sont excitées par des longueurs d'onde de 488 nm ou 532 nm. Les cellules exprimant le gène Luc produisent des enzymes luciférase qui catalysent une réaction avec la luciférine pour produire de la lumière. Le gène lacZ est le gène rapporteur le plus couramment utilisé chez E.coli. Il produit une enzyme β-galactosidase qui fait apparaître la bactérie en bleu lorsqu'elle est cultivée dans un milieu contenant un substrat X-gal.

Gènes rapporteurs pour étudier l’activité du promoteur

Outre l’utilisation de gènes rapporteurs pour étudier l’expression et la localisation des gènes, ils peuvent également contribuer à l’étude de la force des promoteurs. Cela se fait en plaçant un gène rapporteur en aval du promoteur dans un plasmide, puis en l'introduisant dans la cellule de mammifère. L'expression du gène rapporteur surveille l'activité du promoteur. Si l’expression du gène rapporteur est élevée, le promoteur est dit fort ; il est faible si l’expression du gène rapporteur est faible.

Le gène rapporteur, lorsqu'il est placé en aval du promoteur dans un plasmide, aide également à mesurer l'efficacité de la transfection. Le plasmide recombinant est transfecté dans une cellule de mammifère, et son expression montre que la cellule est transfectée avec succès. La quantité de protéine produite par le plasmide recombinant montre son efficacité de transfection.

Gènes rapporteurs

Les gènes rapporteurs sont un type de gène codant pour des protéines qui sont souvent associés à un gène d’intérêt. Une fois à l’intérieur d’une cellule cible, les gènes rapporteurs ...

Education

Enseignement

JoVE Core

Molecular Biology

Biologie moléculaire

Unit 2

Unité 2

Analyzing Gene Expression and Function

Analyse de l'expression et de la fonction géniques

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

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

in vitro mutagenesis

In Vitro Mutagenesis for Gene Analysis

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genetic screens

Using Genetic Screens to Find Disease Genes

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test cross

Test Cross in Genetic Inheritance

A test cross is a genetics tool used to determine the genotype of an organism with a dominant trait. It helps reveal whether the dominant individual is homozygous or heterozygous by crossing it with a recessive individual. The outcome of the cross shows the hidden genotype. If any offspring show the recessive trait, the dominant parent must carry one recessive allele. If all offspring show the dominant trait, the dominant parent is likely homozygous dominant. Test crosses are common in basic...

complementation tests

Using Crosses to Group Mutations

A complementation test uses a simple cross to find out whether two mutations are in the same gene or in different genes. It helps show how mutations are arranged on chromosomes. Edward Lewis first performed this test in the 1940s while studying fruit flies. To do the test, organisms heterozygous for different mutations are crossed pairwise in all combinations. If the mutations are in different genes, they can complement each other. In that case, each mutation provides the missing genetic...

single nucleotide polymorphisms-snps

SNPs as Genetic Markers and Variants

Single nucleotide polymorphisms, or SNPs, are small DNA changes at one specific genomic position. They are the most common type of sequence variation in the human genome. A point mutation counts as an SNP when it appears in more than 1% of the population.

bacterial transformation

Bacterial Transformation in the Lab

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transgenic organisms

Making Transgenic Organisms

Transgenic organisms are living things with DNA added from another organism. A transgene is the inserted gene. Scientists use this method to change an organism’s traits and to study how genes work. To make a transgenic organism, researchers first identify the gene they want to add. They then place that gene into the target organism’s DNA. Once the gene is integrated, the organism can pass it to new cells as it grows. This approach is useful in biology because it helps scientists analyze gene...

reproductive cloning

Cloning an Organism by Reproduction

Reproductive cloning creates a new organism that is genetically the same as the donor. It is different from cloning genes or cells because the goal is to make a whole living individual. This process usually relies on somatic cell nuclear transfer. In this method, the nucleus from a body cell is placed into an egg cell that has had its own nucleus removed. The egg can then develop into an embryo with DNA from the donor cell. Reproductive cloning is used to produce a copy of an organism, such...

crispr

CRISPR Gene Editing Basics

CRISPR is a gene editing tool that helps scientists make targeted changes to DNA. It uses a guide system to find a chosen sequence and a cutting protein to act at that spot. This method is based on a natural defense system found in bacteria. In high school biology, it is often studied as a way to see how genes can be edited with precision. CRISPR is widely used in molecular biology research. It helps scientists explore how genes function and how DNA changes can affect living things.

experimental rnai

RNAi for Gene Silencing and Discovery

RNA interference (RNAi) is a gene-silencing process used to reduce gene expression. It can block transcription or trigger RNA degradation. Scientists first discovered this mechanism in plants in 1998 through Andrew Fire and Craig Mello. RNAi has since been found in many eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. Researchers use it as a practical tool to study gene function. It can show what a gene does without permanently changing the organism’s DNA. <br...

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...

in-situ hybridization

ISH Probes for DNA and RNA Detection

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chromatin immunoprecipitation- chip

ChIP Methods for DNA-Protein Binding

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synthetic biology

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ribosome profiling

Ribosome Profiling for Translation Snapshot

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transgenic plants

Transgenic Plants for Better Crops

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gene therapy

Gene Therapy for Disease Treatment

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epistasis analysis

Gene Interaction Maps in Epistasis

Gene interaction maps in epistasis show how different genes work together to shape a phenotype. In epistasis, the interaction of genes or alleles at different locations affects the final trait. This often happens when one gene masks or interferes with the expression of another gene. Epistasis is common when genes are part of the same biochemical pathway. In that case, the expression of one gene can depend on a gene product made earlier in the pathway. Genes that mask or inhibit other genes in...

genome-wide association studies-gwas

Using SNP Patterns to Link Disease Risk

Genome-wide association studies, or GWAS, look for common SNPs, or single nucleotide polymorphisms, that may be linked to disease. Researchers compare how often a SNP appears in people with a disease and in people without it. When a SNP is seen more often in the disease group, it is considered associated with that disease. GWAS uses chi-square analysis to test the chance that an allele is linked to the disease. This approach does not require scientists to first find the target gene behind the...

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

molecular biology

JoVE Core Biologie Moléculaire illustre les processus moléculaires au sein de la cellule grâce à des leçons vidéos claires et concises. Les applications de ces concepts sont ensuite démontrées grâce à des expériences concrètes en laboratoire.

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DNA Microarrays

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Expression Profiling with Microarrays

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Complementary DNA

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RNA-seq

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The ELISA Method

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Transgenic Organisms

Gene Therapy

Reproductive Cloning

CRISPR

Experimental RNAi

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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 ...

In-situ Hybridization

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Obtain, evaluate, and communicate information on how molecular biotechnology may be used in a variety of fields.
EXPLANATION: The goal is to help students appreciate that the basic science knowledge being studied can be manipulated and used to design useful tools for further scientific investigations, medical treatments, agricultural yields, etc. A detailed understanding of any technical procedures is not expected, but rather a "big picture" view of concepts the technology utilizes and the applications the technology is being used for. These technologies may be best investigated along with the standards from LS1 From Molecules to Organisms: Structure and Functions, as the students are learning about DNA and protein. Comparative DNA and protein sequence analysis is also a large part of current phylogenic research and could accompany LS4 Biological Change: Unity and Diversity. Students may also consider the pros and cons of biotechnical applications. Investigation of techniques utilized could come from field trips to witness the use of these techniques in practice, lab exercises, virtual labs, simulation activities, interviews with professionals, etc. Molecular techniques could include: PCR, electrophoresis, restriction enzyme digestion of DNA, DNA sequencing, plasmid-based transformation, transfection, etc. These techniques are used in fields of medicine, agriculture, biomedical engineering, forensic science, etc.

Karyotyping

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Heterochromatin

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Investigate means by which karyotypes are utilized in diagnostic medicine.
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BIO1.ETS2.2

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Analyze scientific and ethical arguments to support the pros and cons of applications of a specific biotechnology technique such as stem cell usage, in vitro fertilization, or genetically modified organisms.
EXPLANATION: The utilization of new technologies in any field of science is dependent on both economic and social factors. In addition to evaluating these factors, scientists must also consider long-term consequences that may not be initially apparent. The emphasis should be on the construction of a rationale argument that supports a position on the use of an application with ethical and social impact. Students should begin to appreciate the differences in ethical values that exist, and recognize that discussion of these values is imperative as knowledge and technology continue to advance, even when resolutions of differences can be rare. After investigation of a specific biotechnology application, students can write a position paper and/or participate in a classroom debate.

BIO1.ETS2.3

Reporter Genes in Cells and Plasmids

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