Name: in vitro mutagenesis for gene analysis
Uploaded: 2021-04-07T12:12:00.000Z
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>]

mutagênese in vitro

In vitro Mutagenesis

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.

Mutagênese 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 Molecular

Unit 2

Unidade 2

Analyzing Gene Expression and Function

Analisando a Expressão e a Função Gênica

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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

JoVE Core Biologia Molecular mostra os processos moleculares da célula através de vídeos de animação claros e concisos. Além disso, nos vídeos de Cientistas em ação, a aplicação desses conceitos é demonstrada por meio de experimentos reais em laboratórios modernos.

In Vitro Mutagenesis for Gene Analysis

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