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

Naukowcy wykorzystują geny reporterowe do określenia, kiedy i gdzie dochodzi do ekspresji interesującego ich genu. Gen reporterowy koduje białko, które można śledzić, takie jak białko o znanej aktywności enzymatycznej lub fluorescencyjne.
Ekspresja genów jest kontrolowana przez sekwencje cis-regulatorowe zlokalizowane przed lub za regionem kodującym gen w DNA.
Wzorzec i czas ekspresji genów można określić, tworząc rekombinowany DNA z genem reporterowym pod kontrolą interesującej nas sekwencji cis-regulatorowej i wprowadzając ją do komórek lub organizmu.
Ten rekombinowany gen jest wprowadzany do różnych typów komórek i zarówno gen będący przedmiotem zainteresowania, jak i gen reporterowy mogą ulegać ekspresji.
Ponieważ gen będący przedmiotem zainteresowania i gen reporterowy mają tę samą sekwencję cis-regulatorową, ulegają ekspresji w tych samych komórkach i w tym samym czasie. Tak więc, monitorując ekspresję genu reporterowego, naukowiec może śledzić, gdzie gen będący przedmiotem zainteresowania ulega ekspresji.
Jednym z dobrze znanych genów reporterowych jest ten, który koduje białko zwane białkiem zielonej fluorescencji - lub GFP.
GFP odkryto w meduzie Aequorea victoria. Białko wytwarza zieloną fluorescencję w świetle ultrafioletowym, co umożliwia naukowcom śledzenie jego lokalizacji w komórce.
Na przykład, aby zbadać ekspresję β-tubuliny w C. elegans, sekwencja kodująca genu β-tubuliny została zastąpiona genem gfp.
Ekspresja genu gfp jest teraz pod kontrolą promotora genu β-tubuliny.
To rekombinowane DNA zostało wprowadzone do robaka za pomocą mikroiniekcji, a gen uległ ekspresji, podobnie jak β-tubulina.
Lokalizacja GFP monitorowano za pomocą mikroskopii fluorescencyjnej. Ponieważ fluorescencję GFP wykazano w neuronach receptora dotyku, jest to prawdopodobnie miejsce, w którym β-tubulina ulega ekspresji.

<ol>
	<li>Soboleski, Mark R., Jason Oaks i William P. Halford. "Białko zielonej fluorescencji jest ilościowym reporterem ekspresji genów w poszczególnych komórkach eukariotycznych". Czasopismo FASEB 19, nr 3 (2005): 1-20.</li>
	<li>Brogan, John, Fang Li, Wenrong Li, Zhimin He, Qian Huang i Chuan-Yuan Li. "Obrazowanie szlaków molekularnych: geny reporterowe". Badania promieniowania 177, nr 4 (2012): 508-513.</li>
	<li>Serganova, Inna, Vladimir Ponomariov i Ronald Blasberg. "Ludzkie geny reporterowe: potencjalne zastosowanie w badaniach klinicznych." Medycyna i biologia nuklearna 34, nr 7 (2007): 791-807.</li>
</ol>

gen reporterowy

Geny reporterowe to rodzaj genu kodującego białko, który często jest przypisany do genu będącego przedmiotem zainteresowania. Po wejściu do komórki docelowej geny reporterowe zwykle wytwarzają rozpoznawalne wizualnie cechy, takie jak fluorescencja i luminescencja, gdy są wyrażane razem z genem będącym przedmiotem zainteresowania. Zatem geny reporterowe „zgłaszają” obecność lub brak interesujących genów w organizmie, określają wzór ekspresji genów lub śledzą fizyczną lokalizację segmentu DNA lub białka w komórce. Powszechnie stosowanymi genami reporterowymi są - GFP (gen białka zielonej fluorescencji), lacZ (gen β-galaktozydazy), RFP (gen białka czerwonej fluorescencji) i Luc (gen lucyferazy). Komórki wyrażające GFP świecą na zielono, gdy są wzbudzone długościami fal od niebieskiego do zakresu ultrafioletu, podczas gdy komórki wyrażające RFP świecą na czerwono po wzbudzeniu długościami fal 488 nm lub 532 nm. Komórki wykazujące ekspresję genu Luc wytwarzają enzymy lucyferazy, które katalizują reakcję z lucyferyną w celu wytworzenia światła. Gen lacZ jest najczęstszym genem reporterowym stosowanym u E.coli. Wytwarza enzym β-galaktozydazę, który powoduje, że bakterie stają się niebieskie, gdy rosną na podłożu zawierającym substrat X-gal. <h4>Geny reporterowe do badania aktywności promotora</h4> Oprócz wykorzystania genów reporterowych w badaniu ekspresji i lokalizacji genów, mogą one również pomóc w badaniu siły promotorów. Odbywa się to poprzez umieszczenie genu reporterowego poniżej promotora w plazmidzie, a następnie wprowadzenie go do komórki ssaczej. Ekspresja genu reporterowego monitoruje aktywność promotora. Jeśli ekspresja genu reporterowego jest wysoka, mówi się, że promotor jest silny; jest słaby, jeśli ekspresja genu reporterowego jest niska. Gen reporterowy, umieszczony poniżej promotora w plazmidzie, również pomaga w pomiarze wydajności transfekcji. Zrekombinowany plazmid transfekuje się komórkę ssaka, a jego ekspresja pokazuje, że komórka została pomyślnie transfekowana. Ilość białka wytwarzanego przez rekombinowany plazmid pokazuje skuteczność transfekcji.

Gen reporterowy

Geny reporterowe to rodzaj genu kodującego białka, który często jest oznaczany jako gen będący przedmiotem zainteresowania. Po wejściu do komórki docelowej geny reporterowe zwykle ...

Education

JoVE Core

Molecular Biology

Biologia molekularna

Unit 2

Jednostka 2

Analyzing Gene Expression and Function

Analiza ekspresji i funkcji genów

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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

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complementation tests

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single nucleotide polymorphisms-snps

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bacterial transformation

Bacterial Transformation in the Lab

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transgenic organisms

Making Transgenic Organisms

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reproductive cloning

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crispr

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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

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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

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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

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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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