Name: knockout mice and gene replacement
Uploaded: 2026-08-26T08:20:04.844Z
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 (Gene Knockouts)

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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TERMINI E CONCETTI CHIAVE

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

what is genetic engineering?

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dna isolation

Steps in DNA Extraction

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recombinant dna technology

Recombinant DNA in Cloning and GMOs

Recombinant DNA is made by joining DNA from different sources in the lab. Scientists often use it to clone genes and study them in cells that are easy to handle, such as bacteria. Organisms that carry recombinant DNA are called genetically modified organisms, or GMOs. This technology gives scientists new tools for research, medicine, and agriculture. To build recombinant DNA, researchers place a gene of interest into a vector, which is a DNA carrier that moves foreign DNA into a host cell. The...

PCR Cycle for DNA Amplification

PCR is a laboratory method for making many copies of a DNA segment. It can turn a small amount of DNA into millions or even billions of copies in just a few hours. The process uses a heat-resistant DNA polymerase inside a machine called a thermocycler. PCR was developed by Kary Mullis in 1983, and he received the 1993 Nobel Prize in Chemistry for it. The method is fast, relatively inexpensive, and precise. It has become useful for molecular cloning, gene mutagenesis, pathogen detection, gene...

complementary dna

From mRNA to cDNA for Gene Studies

Complementary DNA, or cDNA, is made from messenger RNA (mRNA) so scientists can study which genes are active in cells and tissues. Only genes that are transcribed into mRNA are expressed. By extracting mRNA from cells, researchers can compare gene expression across samples. cDNA is produced by reverse transcription. This process copies mRNA into DNA, which is useful because mRNA does not contain introns, or non-coding regions, and other regulatory sequences. As a result, cDNA can help...

antibiotic selection

Antibiotic Resistance in Cloning

Antibiotic resistance genes help scientists find bacteria that took up a plasmid with a gene of interest. In genetic engineering, this resistance is used as a marker, not just as a medical concern. The same process also connects to the growing problem of resistant bacteria in the real world. Antibiotics, such as penicillin, stop bacterial growth or kill bacteria. When bacteria naturally have, or are engineered to carry, an antibiotic resistance gene, the antibiotic no longer works on them.

transgenic organisms

Creating Transgenic Plants and Animals

Transgenic organisms are genetically engineered to carry a transgene, which is a gene from a different species, in their genome. The inserted gene may be a new gene for that organism or a different version of one it already has. Scientists usually make transgenes with recombinant DNA and DNA cloning methods. Researchers use transgenic bacteria, plants, and animals to answer biological questions and to build useful tools. To make a transgenic organism, they first choose a transgenesis method,...

in-vitro mutagenesis (gene knockouts)

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...

crispr

CRISPR-Cas9 Gene Editing in Action

CRISPR-Cas9 is a genome editing tool that lets scientists change DNA at a specific location. It can add, remove, or rearrange genetic material. Researchers study it because it may help treat genetic disorders such as hemophilia and sickle cell anemia. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats, and Cas9 is a CRISPR-associated protein 9. The basic system includes a Cas9 endonuclease, which is an enzyme that cuts DNA, and a small RNA that guides Cas9 to the...

gene therapy

Gene Delivery for Serious Diseases

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embryonic stem cells

How Embryonic Stem Cells Become Specialized

Embryonic stem cells are pluripotent cells that have not yet specialized. Pluripotent means they can give rise to many different cell types in the body. Because of this, they are useful in science and medicine for making cells needed in research or for replacing cells lost to damage or disease. These cells come from the inner cell mass of a blastocyst, which is an early embryo about 3 to 5 days after fertilization in humans. At this stage, the embryo has not yet implanted in the uterus. Human...

adult stem cells

Adult Stem Cells in the Small Intestine

Adult stem cells help maintain tissues by replacing old or damaged cells. These undifferentiated cells divide to make more stem cells or progenitor cells, which then become mature, specialized cells. Small groups of adult stem cells are found in many tissues, including the bone marrow, brain, skin, and gut. The small intestine shows this process clearly. Its lining, called the epithelium, is renewed continuously by adult stem cells. It is also the fastest replaced tissue in the human body,...

induced pluripotent stem cells

Reprogramming Mature Cells into Stem Cells

Reprogramming mature cells into stem cells lets scientists turn specialized cells back into an unspecialized state. These cells are called induced pluripotent stem cells, or iPSCs. Like embryonic stem cells, iPSCs are pluripotent, which means they can form all cell types. To make iPSCs, researchers start with mature cells such as skin fibroblasts or blood cells. The cells are grown in culture, and genes for several transcription factors are delivered with a viral vector. The cells then make...

reproductive cloning

Cloning Animals with SCNT

Reproductive cloning can produce a genetically identical copy of an entire organism. In adult animals, this is usually done with somatic cell nuclear transfer, or SCNT. Another way clones can form is by splitting an early embryo, which is similar to how identical twins arise naturally. In SCNT, an egg cell is taken from an animal and its nucleus is removed. This creates an enucleated egg. A somatic cell, which is any body cell that is not a sex cell, is then taken from the animal to be cloned.

genomics

Genomics and DNA Sequencing

Genomics studies the full set of genetic material in an organism. In humans, that genome includes the DNA carried in 23 pairs of chromosomes in the nucleus, along with mitochondrial DNA. Genomics looks at both coding DNA and non-coding DNA, which helps scientists study how living things are built and how they differ. This field is useful in many areas of biology and medicine. Scientists use genomics to build phylogenetic trees, improve crop productivity and sustainability, help with criminal...

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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