Activator for genes
MSU scientists have learned to control the activity of genes using a viral enzyme
"Scientific Russia" based on the materials of the press service of the Moscow State University
Russian biologists have proposed a new molecular system for controlling gene activity. It is based on an artificially constructed complex based on a viral enzyme capable of attaching to a specific sequence of the target gene and thereby activating it. The development will help to improve the method of gene therapy, which is promising for the treatment of many hereditary diseases, such as hemophilia and immunodeficiency, and will also be useful for basic research and biotechnological production of recombinant proteins. The results of the study, supported by a grant from the Russian Science Foundation (RNF), are published in the journal Cells (Karagyaur et al., A Novel Cre/lox71-Based System for Inducible Expression of Recombinant Proteins and Genome Editing).
In all living cells, genes work with different intensity: some are very active and provide the production of a large amount of protein needed by the body, while others can be "silent" for a long time. Timely and accurate inclusion of the necessary genes and maintenance of their activity allows the body to develop and function properly. At the same time, by controlling their activity, it is possible to study the mechanisms of the development of genetic diseases. In addition, monitoring the work of genes will increase the accuracy of genome editing systems and the effectiveness of a number of gene therapy drugs. However, the currently existing systems of targeted activation of the necessary genes are imperfect: they are difficult to deliver to cells due to their large size, and they are not always specific, that is, they activate several others besides the target gene.
Scientists from Lomonosov Moscow State University (Moscow) proposed a new compact molecular system capable of working as an artificial protein-transcription factor and activating only the necessary genes. As a basis, scientists took the viral enzyme Cre-recombinase, which normally cuts the DNA molecule strictly in a certain place, thus participating in the process of reproduction of the virus. In this case, the enzyme finds the place where the gap needs to be made, according to the sequence of letters-nucleotides in DNA. Upon discovering this "password", the recombinase binds to the DNA molecule.
In this work, the authors used an inactive Cre recombinase, which was unable to cut DNA, but at the same time accurately found the site needed for its planting and bound to it. Biologists attached a fragment of a transcription factor to the recombinase, whose task was to activate genes. As a result, the recombinase delivered the activator strictly to the desired DNA sites, namely to artificial promoters — seats for regulatory proteins. The specificity of the recombinase ensured high accuracy of the entire proposed system.
The scientists tested the proposed method on human renal epithelial tumor cells by activating an artificially introduced green fluorescent protein gene, as well as several other genes, including the coding growth factor, as well as the CRISPR/Cas9 genome editing system. The last gene was selected in order to test whether the proposed system can be used in gene therapy as a way to increase the effectiveness and specificity of CRISPR/Cas9.
After two days, the luminosity of tumor cells increased up to twenty times, and this proved that the fluorescent protein gene was successfully activated. In addition, the authors confirmed that the amount of RNA read from the rest of the studied sequences increased up to 800 times. This confirmed that this development can be used to activate a variety of genes in solving problems of gene therapy, regenerative medicine, biotechnological production and fundamental research.
Fluorescence of human renal epithelial tumor cells before and after activation of the green fluorescent protein gene. Source: Karagyaur et al.
"Our proposed system allows us to control the desired gene and, if necessary, increase its activity hundreds of times, without affecting the work of other DNA sequences in the cell. This will help to increase the efficiency of genome editing methods, and can also be used in gene therapy and biotechnological production. In the future, we plan to improve our development to make it more convenient for large—scale use," says Maxim Karagyaur, Candidate of Biological Sciences, senior researcher at the Institute of Regenerative Medicine of the Moscow State University.
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