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

Os cientistas podem inativar ou nocautear um gene em animais, geralmente ratos, para aprender mais sobre a fun&#xE7;&#xE3;o do gene geralmente substituindo-o com um vetor de segmenta&#xE7;&#xE3;o, um peda&#xE7;o de DNA projetado. O vetor de direcionamento tem sequ&#xEA;ncias que s&#xE3;o hom&#xF3;logas, id&#xEA;nticas, as sequ&#xEA;ncias antes e depois do gene. Geralmente tamb&#xE9;m tem um marcador de sele&#xE7;&#xE3;o positiva como o gene para resist&#xEA;ncia &#xE0; neomicina, NeoR, no meio e um marcador de sele&#xE7;&#xE3;o negativa como o gene para timidina quinase, TK, em uma extremidade.Quando introduzido em c&#xE9;lulas-tronco embrion&#xE1;rias, ele substitui o gene por meio de recombina&#xE7;&#xE3;o hom&#xF3;loga que ocorre naturalmente entre trechos de DNA com sequ&#xEA;ncias semelhantes. As c&#xE9;lulas onde o gene foi corretamente substitu&#xED;do conter&#xE3;o o positivo marcador, mas n&#xE3;o o negativo permitindo que sejam identificadas na cultura. Essas c&#xE9;lulas s&#xE3;o ent&#xE3;o inseridas num embri&#xE3;o de rato e implantado no &#xFA;tero de uma f&#xEA;mea.O rato resultante tem uma combina&#xE7;&#xE3;o de c&#xE9;lulas normais e c&#xE9;lulas com um gene desligado num cromossoma. Esses ratos s&#xE3;o ent&#xE3;o criados para gerar os chamados ratos desativados que s&#xE3;o homozigotos para o desligamento em todas as c&#xE9;lulas.

Hall, Bradford, Advait Limaye e Ashok B Kulkarni. "Visão geral: Geração de camundongos nocaute genéticos." Protocolos atuais em biologia celular / Conselho editorial, Juan S. Bonifacino ... [et al.] CAPÍTULO (setembro de 2009): Unidade-19.1217. [<a href="https://doi.org/10.1002/0471143030.cb1912s44" target="__blank">Fonte</a>]

mutagénese in-vitro

In-vitro Mutagenesis (Gene Knockouts)

Para aprender mais sobre a fun&#xE7;&#xE3;o de um gene, os investigadores podem observar o que acontece quando o gene &#xE9; inativado ou &#x201C;knocked out&#x201D;, criando animais knockout geneticamente modificados. Murganhos knockout t&#xEA;m sido particularmente &#xFA;teis como modelos para doen&#xE7;as humanas como cancro, doen&#xE7;a de Parkinson e diabetes.
 
 <h4>O Processo</h4>
 
 Genes podem ser aleatoriamente eliminados, ou podem ser alvos espec&#xED;ficos. Para realizar o knockout de um gene em particular, uma por&#xE7;&#xE3;o de DNA chamada vector de clonagem &#xE9; criada e usada para substituir o gene normal, inativando-o dessa forma.
 
 Esses vectores de clonagem t&#xEA;m sequ&#xEA;ncias em cada extremidade id&#xEA;nticas&#x2014;ou hom&#xF3;logas&#x2014;&#xE0;s sequ&#xEA;ncias que flanqueiam cada lado do gene de interesse. Essas sequ&#xEA;ncias hom&#xF3;logas permitem que o vector de clonagem substitua o gene atrav&#xE9;s da recombina&#xE7;&#xE3;o hom&#xF3;loga&#x2014;um processo que ocorre naturalmente entre DNA com sequ&#xEA;ncias semelhantes durante a meiose.
 
 O vetor de clonagem &#xE9; introduzido em c&#xE9;lulas estaminais embrion&#xE1;rias de murganho em cultura, usando m&#xE9;todos como eletropora&#xE7;&#xE3;o&#x2014;uso de pulsos el&#xE9;tricos para criar temporariamente poros na membrana celular. Normalmente, para identificar c&#xE9;lulas onde o vector substituiu corretamente o gene, ele &#xE9; desenhado para incluir um marcador de sele&#xE7;&#xE3;o positivo&#x2014;como o gene para resist&#xEA;ncia &#xE0; neomicina (NeoR)&#x2014;entre as regi&#xF5;es hom&#xF3;logas; e um marcador de sele&#xE7;&#xE3;o negativo&#x2014;como o gene da quinase de timidina viral (TK)&#x2014;ap&#xF3;s uma das regi&#xF5;es hom&#xF3;logas.
 
 As c&#xE9;lulas s&#xE3;o expostas &#xE0; neomicina, e somente aquelas que incorporaram o vetor no seu DNA ir&#xE3;o sobreviver porque t&#xEA;m o gene NeoR. Al&#xE9;m disso, as c&#xE9;lulas, onde o vector substituiu o gene alvo atrav&#xE9;s da recombina&#xE7;&#xE3;o hom&#xF3;loga, n&#xE3;o ter&#xE3;o o gene TK, permitindo-lhes sobreviver na presen&#xE7;a do f&#xE1;rmaco ganciclovir. Portanto, a exposi&#xE7;&#xE3;o ao ganciclovir &#xE9; usada para eliminar c&#xE9;lulas que t&#xEA;m o vetor aleatoriamente inserido no seu genoma, porque essas c&#xE9;lulas ter&#xE3;o o gene TK.
 
 As c&#xE9;lulas com o knockout correto do gene s&#xE3;o ent&#xE3;o inseridas em um embri&#xE3;o de murganho, que &#xE9; implantado no &#xFA;tero de uma f&#xEA;mea, onde se desenvolve at&#xE9; o nascimento. O murganho resultantes &#xE9; uma quimera&#x2014;o que significa que &#xE9; composto por uma mistura de c&#xE9;lulas&#x2014;algumas com DNA normal do embri&#xE3;o, e algumas com o gene eliminado em um cromossoma das c&#xE9;lulas transformadas. Estes murganhos s&#xE3;o cruzados, e os descendentes que cont&#xEA;m o gene na sua linha germinativa s&#xE3;o ainda mais cruzados entre si para criar uma linha de murganhos onde cada c&#xE9;lula &#xE9; homozig&#xF3;tica para o knockout. Estes murganhos knockout podem ser ent&#xE3;o usados para estudar a fun&#xE7;&#xE3;o gen&#xE9;tica.

Mutagénese 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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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?

How Scientists Edit DNA for New Traits

Genetic engineering changes an organism’s DNA to add useful traits. Scientists use it in bacteria, plants, animals, and even viruses for research, medicine, agriculture, and industry. It can bring major benefits, but it also raises ethical concerns about humans and the food supply. This work is possible because DNA has the same basic structure and genetic code across life. That shared code lets scientists modify DNA in several ways. One method is CRISPR/Cas9, often called molecular scissors,...

dna isolation

Steps in DNA Extraction

DNA extraction is the process of removing and purifying DNA from cells. It is used in many biotechnology and research applications, including molecular cloning. The main goal is to free DNA from the cell and prepare it for later study or use. The first step is cell lysis, which means breaking cells open. Researchers may use a homogenizer, sonicator, or bead beater to physically lyse the cells. They also often add detergent to chemically disrupt the lipid-based cell membranes. This helps...

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

Gene therapy is a technique used to treat serious disease by adding a gene to a person’s cells. The added gene may be a healthy copy of a mutated gene. It may also be a different gene that inactivates or compensates for the disease-causing gene. One example is severe combined immunodeficiency, or SCID, caused by a mutation in the gene for adenosine deaminase. In that case, a working copy of the gene can be inserted so the patient’s cells make the enzyme again. This can cure the disease in some...

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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