Three whales of biotechnology
Maxim Russo, <url>
February 4 in the framework of the project "Public lectures Polit.Alexander Panchin, Candidate of Biological Sciences, senior researcher at the Institute of Information Transmission Problems of the Russian Academy of Sciences, author of the popular science book "The Sum of Biotechnology", made a speech. His story was devoted to what biotechnologies give us today and what to expect from biotechnologies in the very near future.
Among the directions of modern biotechnology described by Alexander Panchin, three fundamental methods can be distinguished, which have become the basis of many developments right before our eyes. They can be called the three whales of genetic technology.
The first direction is associated with the use of fluorescent proteins. In the 1960s, Japanese scientist Osamu Shimomura, who worked at Princeton, isolated luciferin from small crustaceans (Cypridina hilgendorfii), which allows them to glow. Then, from the body of the jellyfish Aequorea victoria, Shimomura isolated the protein equorin, which provides luminescence in the presence of calcium ions. However, the equorin itself emitted a blue light, and the jellyfish themselves glow green. Continuing his work, Shimomura discovered that this glow is provided by another protein – green fluorescent protein (GFP). It was the discovery of GFP that gave biologists a powerful research tool, although no one suspected it in the early 1960s.
In 1992, Douglas C. Prasher of the Marine Biology Laboratory in Massachusetts cloned a gene encoding GFP. In 1994, Martin Chalfie first applied this protein in vivo by inducing the desired gene into Escherichia coli and the nematode Caenorhabditis elegans. In the future, Roger Tsien improved this protein. For their work on the fluorescent protein, Sikomura, Chalfi and Tsien received the 2008 Nobel Prize in Chemistry.
Alexander Panchin told how scientists later obtained genes that produce fluorescent proteins of a wide variety of colors. They were even able to paint colored pictures in Petri dishes using bacterial cultures with different fluorescent proteins. But, of course, the importance of these proteins is not in the possibility of their use for painting and it was not for this that the Nobel Prize was given. With their help, researchers were able to see the processes taking place in the body, and even control the results of their own actions. This was made possible by genetic engineering. By inserting a gene encoding a fluorescent protein into the right place of the genome, a researcher can determine when and how this part of the genome "works" by the appearance of fluorescence. When a scientist has several different fluorescent proteins at his disposal, they can be introduced into one organism or into one cell and several processes can be observed in parallel. With the help of such multicolored labeling, it is possible to make the cell nucleus, for example, glow red, the cytoskeleton – blue, mitochondria – green, and so on.
A group of scientists from Harvard University in 2007 even proposed a method that allows you to see each individual neuron of the mouse brain. The method is called Brainbow. It consists in the fact that as a result of certain manipulations, each brain cell receives a random set of fluorescent proteins. And, as you know, from a combination of three colors: red, green and blue – you can get any color. As a result, neurons acquire an individual unique color. Now this method is used not only to study the cells of the nervous system of mice, but also on other animals.
The second method, which was discussed in Alexander Panchin's lecture, is the CRISPR/Cas9 genome editing method, which has been gaining popularity lately. The first part of its name means "Clustered Regularly Interspaced Short Palindromic Repeats" – short palindromic repeats regularly arranged in groups. These are fragments of DNA contained in the genome of bacteria. They correspond to the genomes of viruses that once penetrated the ancestors of this bacterium. Having such a library of fragments of viral genomes, a bacterium can identify viruses penetrating into it, just as an antivirus program of a computer recognizes malicious programs from fragments of computer virus code stored in its library. In bacteria, RNA chains are synthesized from viral DNA fragments. RNA interacts on the principle of complementarity with the DNA of new viruses that penetrate the bacterial cell, and then the Cas9 protein comes into play, which breaks the viral DNA at the site of interaction.
Scientists began to use this mechanism to edit genomes. They synthesize RNAs complementary to the place of the genome where the chain should be cut, and inject them into cells together with the Cas9 protein. More precisely, DNA constructs encoding both are introduced into the cell. As a result, the RNA will point to the place where it should be cut, and the protein will be cut. Making a break in one of the two strands of double–stranded genomic DNA greatly increases the likelihood of homologous recombination, a process in which homologous chromosomes exchange homologous fragments. If a donor DNA carrying the desired gene variant is introduced into a cell, and then a gap is inserted into the right place using the CRISPR/ Cas9 system, then a chromosome with the desired gene is formed with a fairly high probability – the cell, filling the gap, will complete the desired sequence according to the proposed sample.
The CRISPR/Cas9 system compares favorably with other methods of artificial genome modification in efficiency and safety. To modify the genome of a cell with its help, it is necessary that the system works only for a short time at the very beginning, and then, when the genome is edited, it is no longer necessary. This method of genome editing is constantly being improved. Geneticists are making it more and more accurate, ensuring that the protein cuts the DNA chain strictly in the right place, and also selects protein variants that are easier to deliver to the cell. In the most recent versions of this method, it is no longer Cas9 that is used for cutting, but another protein, FokI.
Currently, research is underway in which the CRISPR/Cas9 system is used, for example, to edit the genome of T-lymphocytes, which will create new methods of combating the human immunodeficiency virus, scientists force the pig's immune system to tolerate human organs developing in the pig embryo and even create malaria mosquitoes in whose body the causative agent of malaria does not survive.
Finally, the third method mentioned in Alexander Panchin's lecture is optogenetics. It is based on the use of proteins that organize the transport of ions through the cell membrane depending on the illumination. Usually these proteins are borrowed from algae, bacteria or archaea, in which they are responsible for light-dependent behavior, for example, for movement in better light (this increases the efficiency of photosynthesis). If such a protein is embedded in the membrane of a neuron, then, depending on the direction of ion transport under the influence of light, it can prevent the propagation of an electric pulse or vice versa – to start it.
One of these proteins is channelrhodopsin–2 (channelrhodopsin-2, ChR2), taken from algae. In one experiment, he allowed researchers to create false memories in mice by simply shining an optical cable implanted in the brain at specific groups of neurons. During the experiment, the mice were launched into a new chamber, where they received a weak electric shock. At the same time, the c-fos gene, necessary for memory formation, which turns on when an animal enters a new environment, was associated in these mice with the channelorhodopsin-2 gene introduced artificially into their genome. In the future, the mice, getting into the chamber where they were faced with an electric current, showed timidity and moved little, and if they were put in another cell that was not associated with an unpleasant memory, they behaved calmly and actively. But then scientists shone through an optical cable on the neurons of the hippocampus of mice, activating the channelorhodopsin. And the mice, although they were in a safe cell, began to show a sense of fear, "remembering" the electric shocks received.
Another experiment involving photosensitive proteins was related to the restoration of vision. With non–functioning photosensitive retinal cells (rods and cones), the researchers were able to make another group of retinal nerve cells react to light - bipolar cells, which in the usual case only transmit the signal from the rods and cones further. But they were made photosensitive by adding the channelorhodopsin-2 gene to their genome.
Now, according to Alexander Panchin, scientists have paid attention to proteins that are sensitive not to light radiation, but to temperature. Proteins have been found that, in response to temperature changes, open or block the ion channel, regulating the passage of a signal between nerve cells. Thus, along with optogenetics, thermogenetics has now appeared. Moreover, a protein that reacts to a magnetic field has recently been discovered in the human body, so it may also be possible to use it to regulate neurons.
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05.02.2015