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

- [Istruttore] Gli scienziati
possono rendere inattivo

o eliminare un gene negli
animali, comunemente nei topi,

per saperne di pi&#xF9; sulla
funzione di quel gene,

di solito sostituendolo con
un vettore di targeting,

una particella di DNA ingegnerizzato.

Il vettore di targeting
ha sequenze omologhe,

identiche, alle sequenze
del prima e dopo del gene.

Di solito ha anche un indicatore
di selezione positivo,

come il gene della
resistenza alla neomicina,

e NeoR nel mezzo e un indicatore
di selezione negativo,

come il gene della timidina
chinasi, TK, ad una estremit&#xE0;.

Una volta introdotto nelle
cellule staminali embrionali,

sostituisce il gene attraverso
la ricombinazione omologa,

che esiste in natura tra tratti di DNA

con sequenze similari.

Le cellule in cui il gene &#xE8;
stato sostituito correttamente

conterranno l&#x27;indicatore
positivo, ma non il negativo

questo permette la loro
identificazione in coltura.

Queste cellule vengono poi
inserite in un embrione di topo

e trasferite nell&#x27;utero di una femmina.

Il topo avr&#xE0; una combinazione
di cellule normali

e cellule con il gene
knockout su un cromosoma.

Questi topi sono quindi allevati

e definiti topi knockout, sono omozigoti,

il knockout viene eseguito
in tutte le cellule.

Hall, Bradford, Advait Limaye e Ashok B Kulkarni. "Panoramica: generazione di topi knockout genici". Protocolli attuali in biologia cellulare / Comitato editoriale, Juan S. Bonifacino ... [et Al.] CAPITOLO (settembre 2009): Unità-19.1217. [<a href="https://doi.org/10.1002/0471143030.cb1912s44" target="__blank">Sorgente</a>]

mutagenesi in vitro

In vitro Mutagenesis

Per saperne di pi&#xF9; sulla funzione di un gene, i ricercatori possono osservare cosa succede quando il gene viene inattivato o &#x22;eliminato&#x22;, creando animali geneticamente modificati. I topi Knockout sono stati particolarmente utili come modelli per malattie umane come il cancro, il morbo di Parkinson e il diabete.

<h4>Il processo</h4>

I geni possono essere eliminati casualmente, o geni specifici possono essere presi di mira. Per eliminare un particolare gene, un pezzo di DNA ingegnerizzato chiamato vettore di puntamento viene utilizzato per sostituire il gene normale, inattivandolo in tal modo.

I vettori di &#x22;targeting&#x22; (puntamento) hanno sequenze su ogni estremit&#xE0; identiche, o omologhe, alle sequenze che fiancheggiano ogni lato del gene di interesse. Queste sequenze omologhe permettono al vettore di targeting di sostituire il gene attraverso la ricombinazione omologa, un processo che si verifica naturalmente tra il DNA con sequenze simili durante la meiosi.

Il vettore di targeting viene introdotto nelle cellule staminali embrionali del topo in coltura, utilizzando metodi come l&#x27;elettroporazione, usando impulsi elettrici per creare temporaneamente pori nella membrana cellulare. Tipicamente, per identificare le cellule in cui il vettore ha sostituito correttamente il gene, &#xE8; progettato per includere un marcatore di selezione positiva, come il gene per la resistenza alla neomicina (NeoR),tra le regioni omologhe; e un marcatore di selezione negativo, come il gene della tiofina virale (TK), dopo una delle regioni omologhe.

Le cellule sono esposte alla neomicina, e solo quelle che hanno incorporato il vettore nel loro DNA sopravviveranno perch&#xE9; hanno il gene NeoR. Inoltre, le cellule, dove il vettore ha sostituito il gene bersaglio attraverso la ricombinazione omologa, non avranno il gene TK, permettendo loro di sopravvivere in presenza del farmaco ganciclovir. Pertanto, l&#x27;esposizione al ganciclovir viene utilizzata per eliminare le cellule che hanno il vettore inserito casualmente nel loro genoma, perch&#xE9; queste cellule avranno il gene TK.

Le cellule con il gene correttamente eliminato vengono quindi inserite in un embrione di topo, che viene impiantato nell&#x27;utero di una femmina, dove si sviluppa fino alla nascita. Il topo risultante &#xE8; una chimera, che significa che &#xE8; composto da una miscela di cellule, alcune con DNA normale dall&#x27;embrione, e alcune con il gene eliminato su un cromosoma dalle cellule ingegnerizzate. Questi topi sono allevati, e la prole contenente il gene nella loro linea germinale sono ulteriormente incrociati per creare una linea di topi in cui ogni cellula &#xE8; omozicapra per il knockout. Questi topi knockout possono quindi essere utilizzati per studiare la funzione genica.

Mutagenesi 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

Didattica

JoVE Core

Molecular Biology

Biologia Molecolare

Unit 2

Unità 2

Analyzing Gene Expression and Function

Analisi dell'espressione e della funzione genica

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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...

genetic screens

Using Genetic Screens to Find Disease Genes

Genetic screens are tools used to find genes and mutations linked to specific traits. They are also used to identify people or groups at risk for genetic diseases. That can support early intervention, targeted therapy, and reproductive choices. Forward genetic screens, also called classical screens, start with random DNA changes in an organism. Researchers may use radiation, mutagens, or added bases to create mutations that lead to visible phenotype changes. The mutant is then inbred so...

test cross

Test Cross in Genetic Inheritance

A test cross is a genetics tool used to determine the genotype of an organism with a dominant trait. It helps reveal whether the dominant individual is homozygous or heterozygous by crossing it with a recessive individual. The outcome of the cross shows the hidden genotype. If any offspring show the recessive trait, the dominant parent must carry one recessive allele. If all offspring show the dominant trait, the dominant parent is likely homozygous dominant. Test crosses are common in basic...

complementation tests

Using Crosses to Group Mutations

A complementation test uses a simple cross to find out whether two mutations are in the same gene or in different genes. It helps show how mutations are arranged on chromosomes. Edward Lewis first performed this test in the 1940s while studying fruit flies. To do the test, organisms heterozygous for different mutations are crossed pairwise in all combinations. If the mutations are in different genes, they can complement each other. In that case, each mutation provides the missing genetic...

single nucleotide polymorphisms-snps

SNPs as Genetic Markers and Variants

Single nucleotide polymorphisms, or SNPs, are small DNA changes at one specific genomic position. They are the most common type of sequence variation in the human genome. A point mutation counts as an SNP when it appears in more than 1% of the population.

bacterial transformation

Bacterial Transformation in the Lab

Bacterial transformation is a lab method used to move new DNA into bacteria. Scientists use it to change how bacteria behave or to help them carry a gene of interest. In high school biology, this process is often shown with plasmids, which are small circular DNA molecules. During transformation, bacteria take up DNA from their surroundings. The new DNA can then be used by the cell, depending on the experiment. This technique is an important part of genetic engineering and is widely used in...

transgenic organisms

Making Transgenic Organisms

Transgenic organisms are living things with DNA added from another organism. A transgene is the inserted gene. Scientists use this method to change an organism’s traits and to study how genes work. To make a transgenic organism, researchers first identify the gene they want to add. They then place that gene into the target organism’s DNA. Once the gene is integrated, the organism can pass it to new cells as it grows. This approach is useful in biology because it helps scientists analyze gene...

reproductive cloning

Cloning an Organism by Reproduction

Reproductive cloning creates a new organism that is genetically the same as the donor. It is different from cloning genes or cells because the goal is to make a whole living individual. This process usually relies on somatic cell nuclear transfer. In this method, the nucleus from a body cell is placed into an egg cell that has had its own nucleus removed. The egg can then develop into an embryo with DNA from the donor cell. Reproductive cloning is used to produce a copy of an organism, such...

crispr

CRISPR Gene Editing Basics

CRISPR is a gene editing tool that helps scientists make targeted changes to DNA. It uses a guide system to find a chosen sequence and a cutting protein to act at that spot. This method is based on a natural defense system found in bacteria. In high school biology, it is often studied as a way to see how genes can be edited with precision. CRISPR is widely used in molecular biology research. It helps scientists explore how genes function and how DNA changes can affect living things.

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 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...

in-situ hybridization

ISH Probes for DNA and RNA Detection

In situ hybridization (ISH) uses labeled probes to detect and localize specific DNA or RNA molecules in cells, tissues, or tissue sections. The probe is a complementary strand of DNA or RNA that binds to a matching nucleotide sequence in the sample. Since its first use in 1969 for nucleic acid studies, ISH has become an important tool in research and in clinical diagnosis. Several probe types can be used in ISH. These include single-stranded DNA probes, double-stranded DNA probes, antisense...

chromatin immunoprecipitation- chip

ChIP Methods for DNA-Protein Binding

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation. ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes...

synthetic biology

Synthetic Biology in Crops and Cells

Synthetic biology combines engineering, molecular biology, cell biology, and systems biology to redesign living systems. Scientists use it to remodel organisms from nature or to build new synthetic organisms. These tools can support protein or enzyme production, bioremediation, the production of useful macromolecules, and the addition of helpful traits to crops. One well-known example is golden rice. This genetically modified rice plant makes grains rich in β-carotene, which is a precursor of...

ribosome profiling

Ribosome Profiling for Translation Snapshot

Ribosome profiling is a deep sequencing method that gives a snapshot of active translation in a cell. It sequences the mRNAs protected by ribosomes. This shows which messages are being read to make proteins at a specific moment. The method has several uses in biology. It can track translation in a living organ or tissue type. It also helps measure new protein synthesis levels. Researchers use it to find translated open reading frames, which are stretches of RNA that can make protein. It can...

transgenic plants

Transgenic Plants for Better Crops

Transgenic plants are crops that have been changed with recombinant DNA technology, also called transgenesis. This method adds a foreign gene or removes a harmful one from an organism. The result is a genetically modified organism. The first transgenic plant was a tobacco plant made in 1983, and it was resistant to tobacco mosaic virus. Since then, transgenic plants have been developed for many uses in agriculture, horticulture, and ornamental plants. Some are designed to resist pests or...

gene therapy

Gene Therapy for Disease Treatment

Gene therapy is a treatment approach that changes gene activity to help address disease. It focuses on the genetic cause of a problem rather than only easing symptoms. In this way, it is used in disease intervention. One common strategy is to replace a faulty gene with a normal copy. Another strategy is to inactivate a gene that is causing harm. Gene therapy can also introduce a new gene to help cells work more normally.

epistasis analysis

Gene Interaction Maps in Epistasis

Gene interaction maps in epistasis show how different genes work together to shape a phenotype. In epistasis, the interaction of genes or alleles at different locations affects the final trait. This often happens when one gene masks or interferes with the expression of another gene. Epistasis is common when genes are part of the same biochemical pathway. In that case, the expression of one gene can depend on a gene product made earlier in the pathway. Genes that mask or inhibit other genes in...

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 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

Le video animazioni di JoVE Core Molecular Biology, organizzate in video lezioni, illustrano i processi molecolari cellulari in maniera chiara e concisa. Inoltre, nei video di "scienziati in azione", i concetti scientifici vengono applicati e dimostrati da ricercatori attraverso esperimenti in laboratorio.

In Vitro Mutagenesis for Gene Analysis

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