Mice to study coronavirus
Transgenic mice and COVID-19
Maxim Rousseau, Polit.roo
Scientists at the Institute of Gene Biology of the Russian Academy of Sciences have created transgenic mice that can be infected with the SARS-CoV-2 coronavirus. The experiment confirmed that mice get sick with COVID-19 and, accordingly, can be used as a safe tool for finding medicines and creating vaccines against coronavirus infection.
The development of means to combat COVID-19 is significantly complicated by the fact that scientists do not have laboratory animals suitable for this purpose. Cases of infection with the virus of cats, dogs, ferrets, bats, monkeys, minks, cougars, snow leopards, tigers and shrews have been recorded. The clinical picture of minks and ferrets differs significantly from the manifestations of the disease in humans, they most often have a gastrointestinal type of disease. Dogs and cats, as well as monkeys, cannot be used en masse, and without this it is impossible to identify, for example, rare variants of the course of the disease or side effects of vaccines. The most convenient option would be traditional laboratory animals – rats or mice, but they are not susceptible to infection.
"Mice are very convenient animals for laboratory research. And not only because they eat little (about six grams of feed per day) and multiply quickly (pregnancy lasts 21 days, puberty occurs about a month). The fact is that mice have been studied far and wide. The features of the immune response have been studied, the distribution of drugs in tissues has been studied, the genome has been literally read, the role of specific polymorphisms is known. You can even choose a mouse line with certain properties (with immunodeficiency, obesity, etc.). Due to the fact that clean lines are derived, the control group will always be the control group," explains one of the authors of the study, Alexandra Bruter, an employee of the Institute of Gene Biology of the Russian Academy of Sciences.
If there are no such animals, they need to be created. To do this, you need to supply mice with specific proteins that the SARS-CoV-2 virus needs to attack the body. As is known, the virus, in order to penetrate into a human cell, uses the receptor-binding domain (RBD) of the glycoprotein that makes up the "spikes" on the protein envelope of the virus. This RBD domain connects to the ACE2 protein on the cell membrane. Therefore, in order for mice to become infected with coronavirus infection, the ACE2 protein must also be present in their cell membranes. Mice have their own membrane protein ACE2 (it is designated mACE2, and the human protein is hACE2), but the mouse protein is not similar enough to the human one for the virus to enter the cell with its help.
Another human protein, transmembrane serine protease 2 (TMPRSS2), plays a role in the penetration of the virus. This protein interacts with the "spikes" of the coronavirus and changes them in such a way that they bind better to ACE2. Therefore, the researchers decided to endow their transgenic mice with two proteins at once: both hACE2 and TMPRSS2.
But "teaching" mice to get sick with COVID-19 is not enough. We also need to make it possible to work with such mice. Mice susceptible to coronavirus will become infected with the disease from people working with it, infect each other and die in whole vivariums. Therefore, I would like to be able to turn on and off mouse susceptibility.
To achieve this, the scientists used the viral protein Cre-recombinase. This protein is able to recognize certain DNA sequences (the so-called loxP sites), cut a DNA fragment between two loxP sites and stitch the DNA chain again without the cut fragment. The hACE2 gene was inserted into the mouse genome in a modified form. Each gene has a promoter – a section of DNA that serves as a signal for the RNA polymerase enzyme that it's time to start synthesizing RNA (then this RNA enters the ribosomes, where the corresponding protein is synthesized). But a certain sequence of nucleotides can be placed between the promoter and the rest of the gene, which will be impassable for RNA polymerase, then the synthesis of RNA and further protein from this gene will be impossible. This sequence is called a STOP cassette.
If loxP sites are inserted on both sides of the STOP cassette, and mice are provided with Cre recombinase, then Cre recombinase will cut the STOP cassette from the DNA on command and start the synthesis of the desired protein. Mice will receive the human ACE2 gene at the right moment, and before that they will not be infected with the coronavirus. The signal for Cre-recombinase is the drug tamoxifen. It is given to mice with food for a certain time. Tamoxifen molecules enter cells, where they combine with Cre-recombinase. After that, the recombinase gets the ability to penetrate into the cell nucleus and does its job – cuts out the STOP cassette. And the coronavirus can then infect the mouse.
"An important property of these mice is safety," explains Alexandra Bruter. – They become susceptible to coronavirus only after activation. Before that, they can be kept in a regular vivarium, propagated, transported from place to place and not be afraid that they will get infected from employees. Thanks to the versatility of the STOP cassette, hACE2 expression can be activated at any age in any tissue. Perhaps, thanks to this, it will be possible to find out the causes of a more severe course of the disease in the elderly and the peculiarities of the formation of immunity in people of different ages."
The concept of transgenic mice susceptible to coronavirus, in which the expression of the ACE2 gene depends on Cre recombinase, was proposed by scientists at the Institute of Gene Biology in an article published in May last year. Now they have published a new article in the journal Transgenic Research, where they reported on the successful production of mice with the human ACE2 gene. Mice with the human TMPRSS2 gene are on the approach, and then mice with the genes of both of these proteins. The transgenic mice already obtained were transported from the Institute of Gene Biology to the laboratory of the Vector Scientific Center near Novosibirsk, where they were actually infected with coronavirus and observed the characteristic symptoms of the disease. Now these mice serve as a test system for testing new COVID-19 vaccines.
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