Name: knockout mice and gene replacement
Uploaded: 2026-08-26T08:20:04.844Z
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 mutagenese

In-vitro Mutagenesis (Gene Knockouts)

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.

In vitro mutagenese

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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knockout mice and gene replacement

Knockout mice help researchers study gene function by showing what happens when a gene is turned off. These engineered animals are also useful for modeling human diseases such as cancer, Parkinson’s disease, and diabetes.

A specific gene can be targeted by using a targeting vector, which is a piece of engineered DNA that replaces the normal gene and inactivates it. The vector has matching DNA sequences on both ends. These homologous sequences let the vector swap in for the target gene through homologous recombination, a natural process that happens between similar DNA sequences during meiosis.

Scientists introduce the targeting vector into mouse embryonic stem cells in culture, often by electroporation. Electroporation uses electric pulses to make temporary pores in the cell membrane. The vector is usually built with a positive selection marker, such as the neomycin resistance gene (NeoR), between the homologous regions. It also includes a negative selection marker, such as viral thymidine kinase (TK), after one homologous region.

The cells are treated with neomycin, and only cells that took up the vector survive because they carry NeoR. Cells in which the vector replaced the target gene by homologous recombination survive ganciclovir treatment because they do not have TK. Ganciclovir removes cells in which the vector inserted randomly into the genome, since those cells still carry TK.

The correctly targeted cells are then placed into a mouse embryo. The embryo is implanted into the uterus of a female and allowed to develop until birth. The mouse that results is a chimera, which means it contains a mixture of cells. Some cells come from the original embryo, and some come from the engineered stem cells with one knocked-out chromosome.

These mice are bred, and offspring with the knockout in their germline are crossbred again. This creates a mouse line in which every cell is homozygous for the knockout. Researchers can then use these knockout mice to study the function of the gene.

Knockout Mice and Gene Replacement

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key terms and concepts

See how targeting vectors replace a gene in mouse stem cells, how selection markers work, and how knockout mice are bred for gene function studies.

Knockout Mice and Gene Replacement | JoVE K12

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Knockout Mice and Gene Replacement

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