Name: in vitro mutagenesis for gene analysis
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 29 s
Channel: JoVE

- [Instructeur] Wetenschappers kunnen een gen in dieren, meestal muizen, desactiveren of uitschakelen om meer te leren over de functie van het gen gewoonlijk door het te vervangen door een doelvector een geconstrueerd stuk DNA.
 De doelvector heeft sequenties die homoloog, identiek, zijn aan de sequenties voor en na het gen.
 Meestal heeft het ook een positieve selectiemarker zoals het gen voor neomycine resistentie, NeoR, in het midden en een negatieve selectiemarker zoals het gen voor thymidine kinase, TK, aan een uiteinde.
Wanneer het ingebracht wordt in embryonale stamcellen, vervangt het het gen door homologe recombinatie die vanzelf gebeurt tussen DNA-strengen met gelijkaardige sequenties.
 Cellen waar de genen correct vervangen zijn zulen een positieve marker bevatten, maar geen negatieve, waardoor ze in culturen kunnen ge&#xEF;dentificeerd worden.
 Die cellen worden dan ingebracht in een muisembryo en ge&#xEF;mplanteerd in de baarmoeder van een vrouwtje.
De resulterende muis heeft een combinatie van normale cellen en cellen met een uitgeschakeld gen op &#xE9;&#xE9;n chromosoom.
 Die muizen worden dan gekweekt om zogenaamde &#x27;knockout&#x27; muizen te maken, die homozygoot zijn voor de uitgeschakelde cel in alle cellen.

in vitro mutagenesis

In vitro Mutagenesis

Om meer te weten te komen over de functie van een gen, kunnen onderzoekers observeren wat er gebeurt als het gen wordt ge&#xEF;nactiveerd of &#x2018;uitgeschakeld&#x2019;. door genetisch gemanipuleerde knock-outdieren te cre&#xEB;ren. Knock-outmuizen zijn bijzonder nuttig geweest als model voor ziekten bij de mens, zoals kanker, de ziekte van Parkinson en diabetes.

<h4>Het proces</h4>

Genen kunnen willekeurig worden uitgeschakeld, of specifieke genen kunnen het doelwit zijn. Om een bepaald gen uit te schakelen, wordt een gemanipuleerd stukje DNA, een targetingvector genoemd, gebruikt om het normale gen te vervangen, waardoor het wordt ge&#xEF;nactiveerd.

Targetingvectoren hebben aan elk uiteinde sequenties die identiek &#x2013; of homoloog &#x2013; zijn. aan de sequenties die elke kant van het gen van belang flankeren. Deze homologe sequenties zorgen ervoor dat de targetingvector het gen kan vervangen door middel van homologe recombinatie - een proces dat van nature plaatsvindt tussen DNA met vergelijkbare sequenties tijdens de meiose.

De targetingvector wordt in cultuur gebracht in embryonale stamcellen van muizen, met behulp van methoden zoals elektroporatie &#x2013; het gebruik van elektrische pulsen om tijdelijk pori&#xEB;n in het celmembraan te cre&#xEB;ren. Om cellen te identificeren waar de vector het gen op de juiste manier heeft vervangen, wordt deze doorgaans ontworpen om een positieve selectiemarker &#x2013;zoals het gen voor neomycineresistentie (NeoR) bevinden de homologe gebieden op te nemen; en een negatieve selectiemarker &#x2013; zoals het gen voor viraal thymidinekinase (TK) &#x2013; na een van de homologe gebieden.

De cellen worden blootgesteld aan neomycine en alleen degenen die de vector in hun DNA hebben opgenomen, zullen overleven omdat ze het NeoRgen hebben. Ook zullen cellen waarin de vector het doelgen heeft vervangen door homologe recombinatie niet het TK-gen hebben, waardoor ze kunnen overleven in de aanwezigheid van het medicijn ganciclovir. Daarom wordt blootstelling aan ganciclovir gebruikt om cellen te elimineren waarin de vector willekeurig in hun genoom is ingevoegd, omdat deze cellen het TK-gen zullen hebben.

De cellen waarvan het gen op de juiste manier is uitgeschakeld, worden vervolgens in een muizenembryo ingebracht, dat in de baarmoeder van een vrouwtje wordt ge&#xEF;mplanteerd, waar het zich tot de geboorte ontwikkelt. De resulterende muis is een hersenschim &#x2013; wat betekent dat hij is opgebouwd uit een mengsel van cellen &#x2013; sommige met normaal DNA uit het embryo, en sommige met het gen uitgeschakeld op &#xE9;&#xE9;n chromosoom van de gemanipuleerde cellen. Deze muizen worden gefokt en nakomelingen die het gen in hun kiemlijn bevatten, worden verder gekruist om een muizenlijn te cre&#xEB;ren waarbij elke cel homozygoot is voor de knock-out. Deze knock-outmuizen kunnen vervolgens worden gebruikt om de genfunctie te bestuderen.

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or &#x201C;knocked out,&#x201D; by creating ...

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Education

JoVE Core

Molecular Biology

Moleculaire biologie

Unit 2

Onderdeel 2

Analyzing Gene Expression and Function

Analyse van genexpressie en functie

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

In vitro mutagenese

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 related studies. It supports gene expression and function analysis by showing how a mutation can influence the final outcome of a gene. The technique is an important tool for understanding how genes work in controlled conditions.

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...

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RNAi for Gene Silencing and Discovery

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Gene Therapy for Disease Treatment

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key terms and concepts

Learn how in vitro mutagenesis is used to change DNA and study gene function, helping scientists connect specific mutations to biological effects.

In Vitro Mutagenesis for Gene Analysis | JoVE K12

molecular biology

JoVE Core Molecular Biology showcases the molecular processes of the cell through clear and concise animated video lessons. Additionally, in scientist-in-action videos, these concepts' application is demonstrated through real experiments in modern laboratories.

In Vitro Mutagenesis for Gene Analysis

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