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

Scientists can inactivate or knock out a gene in animals, commonly mice, to learn more about the function of the gene, usually by replacing it with a targeting vector, an engineered piece of DNA.

The targeting vector has sequences that are homologous, identical to the sequences before and after the gene. It usually also has a positive selection marker such as the gene for neomycin resistance NeoR in the middle and a negative selection marker like the gene for thymidine kinase TK at one end.

When introduced into embryonic stem cells it replaces the gene through homologous recombination which occurs naturally between stretches of DNA with similar sequences. Cells where the gene has been correctly replaced will contain the positive marker but not the negative one allowing them to be identified in culture. These cells are then inserted into a mouse embryo and implanted into the uterus of a female.&#xA0;The resulting mouse has a combination of normal cells and cells with the gene knocked out on one chromosome.

These mice are then bred to generate so-called knockout mice that are homozygous for the knockout in all cells.

Hall, Bradford, Advait Limaye i Ashok B Kulkarni. "Przegląd: Pokolenie myszy z nokautem genów". Aktualne protokoły w biologii komórki / Rada redakcyjna, Juan S. Bonifacino ... [i wsp.] ROZDZIAŁ (wrzesień 2009 r.): Jednostka-19.1217. [<a href="https://doi.org/10.1002/0471143030.cb1912s44" target="__blank">Źródło</a>]

mutageneza in vitro

In vitro Mutagenesis

Aby dowiedzie&#x107; si&#x119; wi&#x119;cej na temat funkcji genu, badacze mog&#x105; obserwowa&#x107;, co si&#x119; dzieje, gdy gen zostanie inaktywowany lub &#x201D;znokautowany&#x201D;, tworz&#x105;c genetycznie zmodyfikowane zwierz&#x119;ta z nokautem. Myszy z nokautem okaza&#x142;y si&#x119; szczeg&#xF3;lnie przydatne jako modele chor&#xF3;b ludzkich, takich jak rak, choroba Parkinsona i cukrzyca.

<h4>Proces</h4>

Geny mog&#x105; zosta&#x107; losowo wyeliminowane lub mog&#x105; zosta&#x107; wybrane okre&#x15B;lone geny. Aby wyeliminowa&#x107; konkretny gen, stosuje si&#x119; zmodyfikowany fragment DNA zwany wektorem kieruj&#x105;cym, kt&#xF3;ry zast&#x119;puje normalny gen, inaktywuj&#x105;c go w ten spos&#xF3;b.

Wektory kieruj&#x105;ce maj&#x105; na ka&#x17C;dym ko&#x144;cu sekwencje identyczne&#x2014;lub homologiczne&#x2014;z sekwencjami flankuj&#x105;cymi ka&#x17C;d&#x105; stron&#x119; genu b&#x119;d&#x105;cego przedmiotem zainteresowania. Te homologiczne sekwencje umo&#x17C;liwiaj&#x105; wektorowi kieruj&#x105;cemu zast&#x105;pienie genu poprzez rekombinacj&#x119; homologiczn&#x105; &#x2014; proces, kt&#xF3;ry zachodzi naturalnie mi&#x119;dzy DNA o podobnych sekwencjach podczas mejozy.

Wektor docelowy wprowadza si&#x119; do hodowli embrionalnych kom&#xF3;rek macierzystych myszy, stosuj&#x105;c metody takie jak elektroporacja &#x2014; wykorzystanie impuls&#xF3;w elektrycznych do tymczasowego utworzenia por&#xF3;w w b&#x142;onie kom&#xF3;rkowej.Zazwyczaj, aby zidentyfikowa&#x107; kom&#xF3;rki, w kt&#xF3;rych wektor prawid&#x142;owo zast&#x105;pi&#x142; gen, projektuje si&#x119; go tak, aby zawiera&#x142; marker selekcji pozytywnej &#x2014; taki jak gen oporno&#x15B;ci na neomycyn&#x119; (NeoR) &#x2014; pomi&#x119;dzy regionami homologicznymi; i marker selekcji negatywnej &#x2014; taki jak gen wirusowej kinazy tymidynowej (TK) &#x2014; po jednym z region&#xF3;w homologicznych.

Kom&#xF3;rki s&#x105; wystawione na dzia&#x142;anie neomycyny i tylko te, kt&#xF3;re w&#x142;&#x105;czy&#x142;y wektor do swoDNA, prze&#x17C;yj&#x105;, poniewa&#x17C; maj&#x105; gen NeoR. Ponadto kom&#xF3;rki, w kt&#xF3;rych wektor zast&#x105;pi&#x142; docelowy gen poprzez rekombinacj&#x119; homologiczn&#x105;, nie b&#x119;d&#x105; mia&#x142;y genu TK, umo&#x17C;liwiaj&#x105;cego im prze&#x17C;ycie w obecno&#x15B;ci leku gancyklowiru. Dlatego te&#x17C; nara&#x17C;enie na ganciclovir stosuje si&#x119; w celu wyeliminowania kom&#xF3;rek, do kt&#xF3;rych genomu wprowadzono losowo wektor, poniewa&#x17C; kom&#xF3;rki te b&#x119;d&#x105; mia&#x142;y gen TK.

Kom&#xF3;rki z prawid&#x142;owo usuni&#x119;tym genem wprowadza si&#x119; nast&#x119;pnie do zarodka myszy, kt&#xF3;ry wszczepia si&#x119; do macicy samicy, gdzie rozwija si&#x119; a&#x17C; do porodu. Powsta&#x142;a mysz jest chimer&#x105;&#x2014;co oznacza, &#x17C;e sk&#x142;ada si&#x119; z mieszaniny kom&#xF3;rek &#x2014; niekt&#xF3;rych z normalnym DNA z embrionu, a innych z genem usuni&#x119;tym na jednym chromosomie z zmodyfikowanych kom&#xF3;rek. Te myszy s&#x105; hodowane, a potomstwo zawieraj&#x105;ce gen w linii zarodkowej jest dalej krzy&#x17C;owane w celu stworzenia linii myszy, w kt&#xF3;rej ka&#x17C;da kom&#xF3;rka jest homozygotyczna pod wzgl&#x119;dem nokautu. Te myszy z nokautem mo&#x17C;na nast&#x119;pnie wykorzysta&#x107; do badania funkcji gen&#xF3;w.

Mutageneza in vitro

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

Biologia molekularna

Unit 2

Jednostka 2

Analyzing Gene Expression and Function

Analiza ekspresji i funkcji genów

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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

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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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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