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Mutageneza in vitro
Aby dowiedzieć się więcej na temat funkcji genu, badacze mogą obserwować, co się dzieje, gdy gen zostanie inaktywowany lub ”znokautowany”, tworząc gen…
Scientists can inactivate or knock out a gene in animals, commonly mice, to learn more about the function of the gene, usually by replacing it with a targeting vector, an engineered piece of DNA.
The targeting vector has sequences that are homologous, identical to the sequences before and after the gene. It usually also has a positive selection marker such as the gene for neomycin resistance NeoR in the middle and a negative selection marker like the gene for thymidine kinase TK at one end.
When introduced into embryonic stem cells it replaces the gene through homologous recombination which occurs naturally between stretches of DNA with similar sequences. Cells where the gene has been correctly replaced will contain the positive marker but not the negative one allowing them to be identified in culture. These cells are then inserted into a mouse embryo and implanted into the uterus of a female. The resulting mouse has a combination of normal cells and cells with the gene knocked out on one chromosome.
These mice are then bred to generate so-called knockout mice that are homozygous for the knockout in all cells.
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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.