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

I ricercatori utilizzano i geni reporter per determinare quando e dove viene espresso un gene di interesse. Un gene reporter codifica per una proteina che può essere tracciata, come una proteina con un'attività enzimatica nota o una che è fluorescente.
L'espressione genica è controllata da sequenze cis-regolatorie situate a monte o a valle della regione codificante di un gene nel DNA.
Il modello e la tempistica dell'espressione genica possono essere determinati creando DNA ricombinante con un gene reporter sotto il controllo di una sequenza cis-regolatoria di interesse e introducendolo nelle cellule o in un organismo.
Questo gene ricombinante viene introdotto in diversi tipi di cellule e sia il gene di interesse che il gene reporter possono esprimersi.
Poiché il gene di interesse e il gene reporter hanno la stessa sequenza cis-regolatoria, sono espressi nelle stesse cellule e allo stesso tempo. Quindi, monitorando l'espressione del gene reporter, uno scienziato può tracciare dove viene espresso il gene di interesse.
Uno dei geni reporter più noti è quello che codifica per una proteina chiamata proteina fluorescente verde - o GFP.
La GFP è stata scoperta nella medusa Aequorea victoria. La proteina produce una fluorescenza verde sotto la luce ultravioletta, che consente ai ricercatori di tracciare la sua posizione all'interno di una cellula.
Ad esempio, per studiare l'espressione della β-tubulina in C. elegans, la sequenza codificante del gene della β-tubulina è stata sostituita dal gene gfp.
L'espressione del gene gfp è ora sotto il controllo del promotore del gene della β-tubulina.
Questo DNA ricombinante è stato introdotto nel verme utilizzando una microiniezione e il gene è stato espresso, in modo simile alla β-tubulina.
La posizione della GFP è stata monitorata utilizzando la microscopia a fluorescenza. Poiché la fluorescenza GFP è stata mostrata nei neuroni del recettore tattile, questo è probabilmente un sito in cui viene espressa la β-tubulina.

<ol>
	<li>Soboleski, Mark R., Jason Oaks e William P. Halford. "La proteina fluorescente verde è un reporter quantitativo dell'espressione genica nelle singole cellule eucariotiche." The FASEB journal 19, no. 3 (2005): 1-20.</li>
	<li>Brogan, John, Fang Li, Wenrong Li, Zhimin He, Qian Huang e Chuan-Yuan Li. "Imaging di percorsi molecolari: geni reporter". Ricerca sulle radiazioni 177, n. 4 (2012): 508-513.</li>
	<li>Serganova, Inna, Vladimir Ponomarev e Ronald Blasberg. "Geni reporter umani: potenziale utilizzo negli studi clinici". Medicina nucleare e biologia 34, n. 7 (2007): 791-807.</li>
</ol>

geni reporter

I geni reporter sono un tipo di gene codificante proteine spesso contrassegnati con un gene di interesse. Una volta all'interno di una cellula bersaglio, i geni reporter di solito producono caratteristiche visivamente identificabili come fluorescenza e luminescenza quando espressi insieme al gene di interesse. Pertanto, i geni reporter “segnalano” la presenza o l’assenza di geni di interesse in un organismo, determinano il modello di espressione genetica o tracciano la posizione fisica di un segmento di DNA o di una proteina nella cellula.

I geni reporter comunemente usati sono: GFP (gene della proteina fluorescente verde), lacZ (gene della β-galattosidasi), RFP (gene della proteina fluorescente rossa) e Luc (gene della luciferasi). Le cellule che esprimono GFP si illuminano di verde quando eccitate da lunghezze d'onda dal blu alla gamma dell'ultravioletto, mentre quelle che esprimono RFP si illuminano di rosso quando eccitate da lunghezze d'onda di 488 nm o 532 nm. Le cellule che esprimono il gene Luc producono enzimi luciferasi che catalizzano una reazione con la luciferina per produrre luce. Il gene lacZ è il gene reporter più comune utilizzato nell'E.coli. Produce un enzima β-galattosidasi che fa sì che i batteri appaiano blu quando coltivati in un terreno contenente substrato X-gal.

Geni reporter per studiare l'attività del promotore

Oltre all'uso dei geni reporter nello studio dell'espressione e della localizzazione dei geni, possono anche aiutare nello studio della forza dei promotori. Ciò avviene posizionando un gene reporter a valle del promotore in un plasmide e quindi introducendolo nella cellula di un mammifero. L'espressione del gene reporter monitora l'attività del promotore. Se l'espressione del gene reporter è elevata, si dice che il promotore sia un promotore forte; è debole se l’espressione del gene reporter è bassa.

Anche il gene reporter, quando posizionato a valle del promotore in un plasmide, aiuta a misurare l'efficienza della trasfezione. Il plasmide ricombinante viene trasfettato in una cellula di mammifero e la sua espressione mostra che la cellula è stata trasfettata con successo. La quantità di proteina prodotta dal plasmide ricombinante mostra la sua efficienza di trasfezione.

Geni reporter

I geni reporter sono un tipo di gene codificante proteine che sono spesso etichettati con un gene di interesse. Una volta all'interno di una cellula bersaglio, i geni reporter di solito ...

Education

Didattica

JoVE Core

Molecular Biology

Biologia Molecolare

Unit 2

Unità 2

Analyzing Gene Expression and Function

Analisi dell'espressione e della funzione genica

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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

In vitro mutagenesis is a lab method used to change DNA and study gene function. It helps scientists test how specific DNA changes affect a gene’s activity and the traits it controls. A common approach is to make a known change in a DNA sequence and then examine the result. This can be done by deleting, inserting, or altering selected bases in a gene. The method is useful for linking a DNA change to a biological effect. Researchers use in vitro mutagenesis in molecular biology, genetics, and...

genetic screens

Using Genetic Screens to Find Disease Genes

Genetic screens are tools used to find genes and mutations linked to specific traits. They are also used to identify people or groups at risk for genetic diseases. That can support early intervention, targeted therapy, and reproductive choices. Forward genetic screens, also called classical screens, start with random DNA changes in an organism. Researchers may use radiation, mutagens, or added bases to create mutations that lead to visible phenotype changes. The mutant is then inbred so...

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

Bacterial transformation is a lab method used to move new DNA into bacteria. Scientists use it to change how bacteria behave or to help them carry a gene of interest. In high school biology, this process is often shown with plasmids, which are small circular DNA molecules. During transformation, bacteria take up DNA from their surroundings. The new DNA can then be used by the cell, depending on the experiment. This technique is an important part of genetic engineering and is widely used in...

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

In situ hybridization (ISH) uses labeled probes to detect and localize specific DNA or RNA molecules in cells, tissues, or tissue sections. The probe is a complementary strand of DNA or RNA that binds to a matching nucleotide sequence in the sample. Since its first use in 1969 for nucleic acid studies, ISH has become an important tool in research and in clinical diagnosis. Several probe types can be used in ISH. These include single-stranded DNA probes, double-stranded DNA probes, antisense...

chromatin immunoprecipitation- chip

ChIP Methods for DNA-Protein Binding

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation. ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes...

synthetic biology

Synthetic Biology in Crops and Cells

Synthetic biology combines engineering, molecular biology, cell biology, and systems biology to redesign living systems. Scientists use it to remodel organisms from nature or to build new synthetic organisms. These tools can support protein or enzyme production, bioremediation, the production of useful macromolecules, and the addition of helpful traits to crops. One well-known example is golden rice. This genetically modified rice plant makes grains rich in β-carotene, which is a precursor of...

ribosome profiling

Ribosome Profiling for Translation Snapshot

Ribosome profiling is a deep sequencing method that gives a snapshot of active translation in a cell. It sequences the mRNAs protected by ribosomes. This shows which messages are being read to make proteins at a specific moment. The method has several uses in biology. It can track translation in a living organ or tissue type. It also helps measure new protein synthesis levels. Researchers use it to find translated open reading frames, which are stretches of RNA that can make protein. It can...

transgenic plants

Transgenic Plants for Better Crops

Transgenic plants are crops that have been changed with recombinant DNA technology, also called transgenesis. This method adds a foreign gene or removes a harmful one from an organism. The result is a genetically modified organism. The first transgenic plant was a tobacco plant made in 1983, and it was resistant to tobacco mosaic virus. Since then, transgenic plants have been developed for many uses in agriculture, horticulture, and ornamental plants. Some are designed to resist pests or...

gene therapy

Gene Therapy for Disease Treatment

Gene therapy is a treatment approach that changes gene activity to help address disease. It focuses on the genetic cause of a problem rather than only easing symptoms. In this way, it is used in disease intervention. One common strategy is to replace a faulty gene with a normal copy. Another strategy is to inactivate a gene that is causing harm. Gene therapy can also introduce a new gene to help cells work more normally.

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

Le video animazioni di JoVE Core Molecular Biology, organizzate in video lezioni, illustrano i processi molecolari cellulari in maniera chiara e concisa. Inoltre, nei video di "scienziati in azione", i concetti scientifici vengono applicati e dimostrati da ricercatori attraverso esperimenti in laboratorio.

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Bacterial Transformation: Electroporation

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Expression Profiling with Microarrays

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Complementary DNA

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RNA-seq

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Transgenic Organisms

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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.
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Karyotyping

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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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