Looking for jewels at the other end of the Brainbow

A year ago, I took a course “Mouse genetics; models for human diseases ”held by the International Center for Genetic Engineering and Biotechnology (ICGEB). I have been observing this organization for a long time (since 2014) and when the opportunity arises, I come to them at conferences and workshops. At the moment ICGEB covers 98 countries, of which 65 are members of the organization. Unfortunately, Ukraine is not yet a participating country, but about 2 years ago a collaboration agreement was signed with ICGEB. Cooperation of states with ICGEB promises researchers great prospects, for example, the opportunity to carry out scientific work in the heart of the organization Trieste (Italy) or to take grant participation in courses and conferences. Masters and dissertators of the participating states are given the opportunity to conduct research on the latest equipment.For Ukraine, this is a vital factor, so I look forward to the signing of the agreement between the Ambassador of Ukraine to Italy Yevgeny Perelygin and ICGEB. I note that for scientists from countries that are not collaborators of the organization, the doors are also open. But in this case, you will have to pay the registration fee yourself.

I will return to my story about last year's course. I was overly pleased to be able to participate in the talk “The mouse rodent model (root vole, Microtus oeconomus Pall.) In the ecotoxity bioindication” . Of course, my topic could not compete in all aspects with innovative developments brought by other course participants. And the reports of ICGEB lab employees were generally beyond praise! However, model animals - this is still my topic: after all, I worked for 4 years at the Institute of Molecular Biology and Genetics, simulating all kinds of diseases in small rodents. And also we had a GFP mouse, which is also used as a model animal. Therefore, I, drunk with delight, sat and listened spellbound to the speakers.

I must admit that earlier I didn’t even know about the existence of the German Mouse Clinic in Germany . Dr. Wolfgang Wurst in a simple and accessible form spoke about the secrets of creating lines of transgenic animal models with different human genes (depending on the will of the experimenter). The scientist explained how important it is after receiving the first generation of transgenic animals to responsibly breed the obtained animals according to the relevant zootechnical principles, increase the number of livestock with constant monitoring of the presence of human genes in the resulting offspring. Examples of control include, among other things, common visual characteristics, for example, changes in the animal's coat, tail size, average size, etc. For example, if we model a homozygous individual for the MSTN gene, then the mouse will be significantly larger than the wild type). His clinic also uses ultrasound and X-ray machines for mice. For example, Paget's disease is characterized by impaired bone remodeling, which leads to enlargement and deformation of bones. On the x-ray characteristic changes will be visible. This disease is caused by a mutation in the SQSTM1 and RANK genes . Dr. Wolfgant Wurst also mentioned, as a reference, the Brainbow model .

The Brainbow model aims to map the interconnections of neurons in the brain. In order to create such a model, it is necessary to knock out those genes that are of interest to the biotechnologist using the so-called Cre-LoxP system tool . This system consists of Crerecombinases and LoxP sites (DNA sequence consisting of 34 nucleotide pairs). This system was spied on and borrowed from nature, namely the P1 phage. In the P1 phage, this system allows the normal life cycle of this virus with the insertion of the phage into the bacterial genome. In the experiment, scientists introduce a cassette with the genes of 4 fluorescent proteins and six LoxP sites ( 3 pairs : 1, 2 and 3) between the genes so that the pairs are directed in the same direction. This way of introducing LoxP sites provides a cut of the area between identical LoxP sites. Provides this enzyme Cre- recombinase. So, the enzyme Cre-recombinase folds the DNA molecule in such a way as to line up identical LoxP sites opposite each other, and produces a cross between these regions of the DNA molecule. This leads to the fact that the area that was between the two LoxP- sites is enclosed in a loop and subsequently cut out of the molecule. The parcels that were to the right and left of the LoxP sites are linked together. Therefore, such a system is called knockout - as it turns off certain genes. But the Brainbow model is not just a knockout , but Conditional Knockou t, that is, a knockout under certain conditions. For this, Cre- recombinase is activated only in hippocampal cells.... In order for Cre- recombinase to be triggered only in specific cells, a special promoter is selected, in this case the THY1 gene promoter . In this design, there are four parts at once that code for fluorescent proteins: orange, red, yellow and blue. If the recombination passes through the sites of the first pair, then the orange fluorescent protein will be removed from the construct, and the red fluorescent protein will combine with the promoter. If recombination occurs at the sites of the second pair, the orange and red fluorescent proteins will be removed, and the promoter will combine with yellow. Well, the third option - orange, red and yellow will be removed, and only blue will remain. This leads to the fact that each cell is painted in its own color. Due to the combinations of different fluorochromes and colors, about 70individual color combinations that are distinguishable by a computer.

From such cells, where successful homologous recombination has occurred, transgenic animals can be obtained. When crossed with a strain of mice with the Cre-recombinase gene, the offspring were obtained carrying both the Cre- recombinase gene and the "multicolor" cassette. In such mice, recombination will occur and the neurons will be stained - it will be easy for scientists to trace where the processes of nerve cells begin and end, and, therefore, it is possible to track the interconnections of the neurons in the brain.

The Brainbow model really can be called a reference. She is very graceful. By the way, this week I was flipping through the news of the Bogomolets Institute and saw the video of Medexpertiza - Channel 5. The video is devoted to the study of pain processes, sensations of its intensity and overcoming without analgesics. Yaroslav Andrianov, a postgraduate student at the Institute of Physiology of the National Academy of Sciences of Ukraine, talks about his research, and one of his computer screens illustrates the dentate gyrus in the mouse hippocampus, stained by the Brainbow method. Figure from TA Weissman et al. Generating and imaging multicolor Braibow mice . Very interesting and informative video. Probably, this video plot prompted me to write this note.

PS good link to Cre-LoxP recombination on the internet

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