Выключение генов: методы и применение в исследованиях.

Genetic technique

Gene knockouts (also known as gene deletion or gene inactivation) are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination, CRISPR Cas9, and TALENs. One of the main advantages of gene knockouts is that they allow researchers to study the function of a specific gene in vivo, and to understand the role of the gene in normal development and physiology as well as in the pathology of diseases. By studying the phenotype of the organism with the knocked out gene, researchers can gain insights into the biological processes that the gene is involved in. There are two main types of gene knockouts: complete and conditional. A complete gene knockout permanently inactivates the gene, while a conditional gene knockout allows for the gene to be turned off and on at specific times or in specific tissues. Conditional knockouts are particularly useful for studying developmental processes and for understanding the role of a gene in specific cell types or tissues. Gene knockouts have been widely used in many different organisms, including bacteria, yeast, fruit flies, zebrafish, and mice. In mice, gene knockouts are commonly used to study the function of specific genes in development, physiology, and cancer research. The use of gene knockouts in mouse models has been particularly valuable in the study of human diseases. For example, gene knockouts in mice have been used to study the role of specific genes in cancer, neurological disorders, immune disorders, and metabolic disorders. However, gene knockouts also have some limitations. For example, the loss of a single gene may not fully mimic the effects of a genetic disorder, and the knockouts may have unintended effects on other genes or pathways. Additionally, gene knockouts are not always a good model for human disease as the mouse genome is not identical to the human genome, and mouse physiology is different from human physiology. The KO technique is essentially the opposite of a gene knock in. Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe three or four knocked out genes, respectively. However, one needs to distinguish between heterozygous and homozygous KOs. In the former, only one of two gene copies (alleles) is knocked out, in the latter both are knocked out.

Knockouts are accomplished through a variety of techniques. Originally, naturally occurring mutations were identified and then gene loss or inactivation had to be established by DNA sequencing or other methods.

Gene knockout by mutation is commonly carried out in bacteria. An early instance of the use of this technique in Escherichia coli was published in 1989 by Hamilton, et al. In this experiment, two sequential recombinations were used to delete the gene. This work established the feasibility of removing or replacing a functional gene in bacteria. That method has since been developed for other organisms, particularly research animals, like mice. Knockout mice are commonly used to study genes with human equivalents that may have significance for disease. An example of a study using knockout mice is an investigation of the roles of Xirp proteins in Sudden Unexplained Nocturnal Death Syndrome (SUNDS) and Brugada Syndrome in the Chinese Han Population.

For gene knockout investigations, RNA interference (RNAi), a more recent method, also known as gene silencing, has gained popularity. In RNA interference (RNAi), messenger RNA for a particular gene is inactivated using small interfering RNA (siRNA) or short hairpin RNA (shRNA). This effectively stops the gene from being expressed. Oncogenes like Bcl 2 and p53, as well as genes linked to neurological disease, genetic disorders, and viral infections, have all been targeted for gene silencing utilizing RNA interference (RNAi).

Homologous recombination is the exchange of genes between two DNA strands that include extensive regions of base sequences that are identical to one another. In eukaryotic species, bacteria, and some viruses, homologous recombination happens spontaneously and is a useful tool in genetically engineered. Homologous recombination, which takes place during meiosis in eukaryotes, is essential for the repair of double stranded DNA breaks and promotes genetic variation by allowing the movement of genetic information during chromosomal crossing. Homologous recombination, a key DNA repair mechanism in bacteria, enables the insertion of genetic material acquired through horizontal transfer of genes and transformation into DNA. Homologous recombination in viruses influences the course of viral evolution. Homologous recombination, a type of gene targeting used in genetic engineering, involves the introduction of an engineered mutation into a particular gene in order to learn more about the function of that gene. This method involves inserting foreign DNA into a cell that has a sequence similar to the target gene while being flanked by sequences that are the same upstream and downstream of the target gene. The target gene's DNA is substituted with the foreign DNA sequence during replication when the cell detects the similar flanking regions as homologues. The target gene is "knocked out" by the exchange. By using this technique to target particular alleles in embryonic stem cells in mice, it is possible to create knockout mice. With the aid of gene targeting, numerous mouse genes have been shut down, leading to the creation of hundreds of distinct mouse models of various human diseases, such as cancer, diabetes, cardiovascular diseases, and neurological disorders. Mario Capecchi, Sir Martin J. Evans, and Oliver Smithies performed groundbreaking research on homologous recombination in mouse stem cells, and they shared the 2007 Nobel Prize in Physiology or Medicine for their findings. Traditionally, homologous recombination was the main method for causing a gene knockout. This method involves creating a DNA construct containing the desired mutation. For knockout purposes, this typically involves a drug resistance marker in place of the desired knockout gene. The construct will also contain a minimum of 2kb of homology to the target sequence. The construct can be delivered to stem cells either through microinjection or electroporation. This method then relies on the cell's own repair mechanisms to recombine the DNA construct into the existing DNA. This results in the sequence of the gene being altered, and most cases the gene will be translated into a nonfunctional protein, if it is translated at all. However, this is an inefficient process, as homologous recombination accounts for only 10−2 to 10−3 of DNA integrations. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene. This process is more efficient than homologous recombination, and therefore can be more easily used to create biallelic knockouts. The DNA binding region consists of amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene. The coupled Cas9 will cause a double stranded break in the DNA.

Gene knock in is similar to gene knockout, but it replaces a gene with another instead of deleting it.

A conditional gene knockout allows gene deletion in a tissue in a tissue specific manner. This is required in place of a gene knockout if the null mutation would lead to embryonic death, or a specific tissue or cell type is of specific interest. This is done by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ line via the same mechanism as a knockout. This germ line can then be crossed to another germline containing Cre recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites. Genes not involved in early development have been effectively studied using knockout approaches that use gene deletion. However, it is typically not possible to knock off genes that are active during early development without the organism suffering a fatal outcome. One method around this is conditional knockout. Utilizing a site specific recombinase called Cre, the original conditional knockout technique recombined short target sequences known as LoxP. Since then, other recombinases have been created and employed in conditional knockout experiments.

Knockouts are primarily used to understand the role of a specific gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. Knockout organisms are also used as screening tools in the development of drugs, to target specific biological processes or deficiencies by using a specific knockout, or to understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae.