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Mutagénese In-vitro
Para aprender mais sobre a função de um gene, os investigadores podem observar o que acontece quando o gene é inativado ou “knocked out”, criando anim…
Os cientistas podem inativar ou nocautear um gene em animais, geralmente ratos, para aprender mais sobre a função do gene geralmente substituindo-o com um vetor de segmentação, um pedaço de DNA projetado. O vetor de direcionamento tem sequências que são homólogas, idênticas, as sequências antes e depois do gene. Geralmente também tem um marcador de seleção positiva como o gene para resistência à neomicina, NeoR, no meio e um marcador de seleção negativa como o gene para timidina quinase, TK, em uma extremidade.
Quando introduzido em células-tronco embrionárias, ele substitui o gene por meio de recombinação homóloga que ocorre naturalmente entre trechos de DNA com sequências semelhantes. As células onde o gene foi corretamente substituído conterão o positivo marcador, mas não o negativo permitindo que sejam identificadas na cultura. Essas células são então inseridas num embrião de rato e implantado no útero de uma fêmea.
O rato resultante tem uma combinação de células normais e células com um gene desligado num cromossoma. Esses ratos são então criados para gerar os chamados ratos desativados que são homozigotos para o desligamento em todas as células.
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Q1: What is a targeting vector and how does it work in gene knockout?
A targeting vector is an engineered piece of DNA designed to replace a specific gene through homologous recombination. It contains sequences identical to the DNA flanking the target gene, allowing it to recognize and replace the correct location. The vector also includes a positive selection marker like neomycin resistance and a negative selection marker like thymidine kinase to identify successful replacements in embryonic stem cells.
Q2: How does homologous recombination enable gene replacement in knockout mice?
Homologous recombination is a natural process where DNA sequences with identical or homologous regions exchange genetic material. When a targeting vector containing homologous sequences is introduced into embryonic stem cells, the cell's machinery recognizes the matching sequences and replaces the target gene with the vector. This precise mechanism ensures the correct gene is inactivated rather than random DNA insertion.
Q3: Why are selection markers used in targeting vectors?
Selection markers identify cells where the targeting vector has successfully replaced the gene. The positive marker, such as neomycin resistance, allows only cells containing the vector to survive antibiotic exposure. The negative marker, like thymidine kinase, is present only in cells with random vector insertion, enabling researchers to eliminate these unwanted cells using ganciclovir and isolate correctly targeted cells.
Q4: What is a chimera mouse and why is it an intermediate step?
A chimera mouse is composed of a mixture of cells—some with normal DNA and some with the knocked-out gene on one chromosome. It results from inserting engineered embryonic stem cells into a mouse embryo. Chimera mice are intermediate because they are heterozygous for the knockout; they must be bred with each other to produce offspring where every cell is homozygous for the knockout.
Q5: How are knockout mice generated from chimera mice?
Chimera mice are bred together, and offspring carrying the knockout gene in their germline are selected. These mice are further crossbred to create a line where every cell is homozygous for the knockout, meaning both copies of the gene are inactivated. This multi-generational breeding process ensures consistent knockout expression across all tissues for reliable research studies.
Q6: What are common applications of knockout mice in disease research?
Knockout mice serve as valuable models for understanding human diseases by observing what happens when specific genes are inactivated. They have been particularly useful for studying cancer, Parkinson's disease, and diabetes. By comparing knockout mice to normal mice, researchers can determine how the absent gene contributes to disease development and progression.
Q7: How is the targeting vector introduced into embryonic stem cells?
The targeting vector is introduced into mouse embryonic stem cells using electroporation, a technique that applies electric pulses to temporarily create pores in the cell membrane. This allows the engineered DNA to enter the cells where homologous recombination can occur. Once inside, cells with successful gene replacement are identified using selection markers and then inserted into mouse embryos for development.