Name: knockout mice and gene replacement
Uploaded: 2026-08-26T08:20:04.844Z
Duration: 2 min 29 s
Channel: JoVE

Wissenschaftler k&#xF6;nnen ein Gen in Tieren, im Allgemeinen M&#xE4;usen, inaktivieren oder ausschalten, um mehr &#xFC;ber die Funktion des Gens zu erfahren.
 In der Regel wird es durch einen Targeting-Vektor, ein gentechnisch hergestelltes St&#xFC;ck DNA, ersetzt.
 Der Targeting-Vektor weist Sequenzen auf, die homolog und identisch zu den Sequenzen vor und nach dem Gen sind.
 Es hat normalerweise auch einen positiven Selektionsmarker wie das Gen f&#xFC;r Neomycinresistenz, NeoR, in der Mitte und einen negativen Selektionsmarker wie das Gen f&#xFC;r Thymidinkinase, TK, an einem Ende.
 Wenn es in embryonale Stammzellen eingef&#xFC;hrt wird, ersetzt es das Gen durch homologe Rekombination, die auf nat&#xFC;rliche Weise zwischen DNA-Abschnitten mit &#xE4;hnlichen Sequenzen auftritt.
 Zellen, bei denen das Gen korrekt ersetzt wurde, enthalten den positiven, nicht aber den negativen Marker, sodass sie in Kultur identifiziert werden k&#xF6;nnen.
 Diese Zellen werden dann in einen Mausembryo eingef&#xFC;hrt und in die Geb&#xE4;rmutter eines Weibchens implantiert.
 Die resultierende Maus hat eine Kombination aus normalen Zellen und Zellen, bei denen das Gen auf einem Chromosom ausgeschaltet ist (Gen-Knockout).
 Diese M&#xE4;use werden dann gez&#xFC;chtet, um sogenannte Knockout-M&#xE4;use zu erzeugen, die f&#xFC;r den Knockout in allen Zellen homozygot sind.

Hall, Bradford, Advait Limaye und Ashok B Kulkarni. "Überblick: Erzeugung von Gen-Knockout-Mäusen." Aktuelle Protokolle in der Zellbiologie / Redaktion, Juan S. Bonifacino ... [et al.] KAPITEL (September 2009): Einheit-19.1217. [<a href="https://doi.org/10.1002/0471143030.cb1912s44" target="__blank">Quelle</a>]

in-vitro-mutagenese

In-vitro Mutagenesis (Gene Knockouts)

Um mehr &#xFC;ber die Funktion eines Gens zu erfahren, k&#xF6;nnen Forscher beobachten, was passiert, wenn das Gen inaktiviert oder entfernt (knock out) durch die Schaffung genetisch manipulierter &#x22;Knockout-Tiere&#x201C;. Knockout-M&#xE4;use erwiesen sich als besonders n&#xFC;tzliches Modell f&#xFC;r menschliche Krankheiten wie Krebs, Parkinson und Diabetes.
 
 <h4>Der Prozess</h4>
 
 Gene k&#xF6;nnen zuf&#xE4;llig ausgeschalten werden, oder es k&#xF6;nnen bestimmte Gene gezielt manipuliert werden. Um ein bestimmtes Gen auszuschalten, wird ein k&#xFC;nstliches St&#xFC;ck DNA, ein sogenannter Targeting-Vektor, verwendet. Dieser ersetzt das normale Gen und inaktiviert es dadurch.
 
 Targeting-Vektoren haben an jedem Ende Sequenzen, die identisch bzw. homolog zu den Sequenzen sind, die die Seiten des gew&#xFC;nschten Gens flankieren. Diese homologen Sequenzen erlauben es dem Zielvektor, das Gen durch homologe Rekombination zu ersetzen. Das ist ein Prozess, der auf nat&#xFC;rliche Weise zwischen DNA mit &#xE4;hnlichen Sequenzen w&#xE4;hrend der Meiose auftritt.
 
 Der Targeting-Vektor wird in kultivierte embryonale Stammzellen der Maus eingef&#xFC;hrt. Dabei werden Methoden wie die Elektroporation, das ist die Verwendung von elektrischen Impulsen zur tempor&#xE4;ren Erzeugung von Poren, in der Zellmembran verwendet. Um Zellen zu identifizieren, bei denen der Vektor das Gen richtig ersetzt hat, enth&#xE4;lt er typischerweise einen positiven Selektionsmarker, wie z.B. das Gen f&#xFC;r Neomycin-Resistenz (NeoR), zwischen den homologen Regionen. Zus&#xE4;tzlich enth&#xE4;lt er nach einer der homologen Regionen, auch einen negativen Selektionsmarker, wie zum Beispiel das Gen f&#xFC;r virale Thymidin-Kinase (TK).
 
 Die Zellen werden dann Neomycin ausgesetzt und nur, die die den Vektor in ihre DNA eingebaut haben, werden &#xFC;berleben, da sie das NeoR-Gen haben. Nur die Zellen, bei denen der Vektor das Zielgen durch homologe Rekombination ersetzt hat, haben kein TK-Gen und k&#xF6;nnen somit in Gegenwart des Medikaments Ganciclovir &#xFC;berleben. Daher wird Ganciclovir verwendet, Zellen zu eliminieren, die den Vektor zuf&#xE4;llig in ihr Genom eingef&#xFC;gt haben, da diese Zellen das TK-Gen haben werden.
 
 Die Zellen mit dem richtig inaktiviertem Gen werden dann in einen Maus-Embryo eingesetzt, der in die Geb&#xE4;rmutter eines Weibchens eingepflanzt wird, wo er sich bis zur Geburt entwickelt. Die resultierende Maus ist eine Chim&#xE4;re. Das hei&#xDF;t, dass sie vermischte Zellen besitzt. Einige dieser Zellen haben die normale DNA von dem Embryo, w&#xE4;hrend andere das ausgeschaltete Gen auf einem Chromosom aus den manipulierten Zellen besitzen. Diese M&#xE4;use werden gez&#xFC;chtet und die Nachkommen, die das Gen in ihrer Keimbahn enthalten, werden weiter gekreuzt, um eine Linie von M&#xE4;usen zu schaffen, bei der jede Zelle homozygot f&#xFC;r den Knockout ist. Diese Knockout-M&#xE4;use k&#xF6;nnen dann zur Untersuchung der Genfunktion verwendet werden.

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.

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Knockout Mice and Gene Replacement

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