10 July 2014

Pharmaceutical factories on a Brazilian farm

Milk mice and medicinal goats

Yulia Chernaya, "Trinity variant" No. 13-2014

Mice are not the milkiest animals. One mouse gives an average of four drops of milk a day. There are, of course, record holders, whose milk yields reach up to eight drops! But there are also those from which it is barely possible to express one. Moreover, this is a very difficult matter for "milkmaids" and unpleasant for mice (animals have to be milked under general anesthesia using a weak air pump). Nevertheless, the monument to the laboratory mouse was installed next to the Institute of Cytology and Genetics for a reason: it is easier for scientists to deal with small, unpretentious, fast-growing and easily reproducing mice than with more dairy goats, and even more so cows. So the staff of the Laboratory of Developmental Genetics of the Institute of Cytology and Genetics of the SB RAS had to go through the mouse stage on their way from the idea of a new drug that will help patients recover after chemotherapy to goats from whose milk this medicine can be isolated.

The goal of Novosibirsk scientists was to create colony-stimulating factors (granulocyte colony-stimulating factor and granulocyte-macrophage-colony-stimulating factor). These hormones are sometimes called blood-forming growth factors. They play an extremely important role in the treatment of cancer patients. These factors do not directly affect tumor cells, but they stimulate the division of bone marrow stem cells. It is these proteins that influence the creation of white blood cells (leukocytes), platelets and red blood cells (erythrocytes) in our body.

Proteins are the key to treating many diseases. Moreover, if we create a medicine for people based on human protein, it will not cause allergies, addiction, etc. The question arises of how to produce human protein. The simplest option that doctors have come up with for a very long time is to extract these proteins from donated blood. Medicines obtained in this way are very expensive, and a huge amount of blood is required for their production. Another option is to force those organisms to produce human proteins, which in ordinary life is unusual. For example, 90% of the insulin that is so necessary for diabetic patients is produced today thanks to genetically modified microorganisms. A section of the human gene responsible for the production of a specific protein is embedded in the genome of bacteria. Insulin has a fairly simple spatial structure, and bacteria can create it. Unfortunately, most of the proteins that modern medicine needs are much more complex. Bacteria obediently carry out the information about the protein sewn into their genes, but they do not know how to properly modify the resulting protein molecule, so the proteins developed in bacteria are different from human ones.

It is not surprising that scientists have turned their eyes back to mammals. They proposed to create dairy bioreactors: promoters – the "trigger keys" of the genes of dairy cattle (cows, goats, sheep), working only in the mammary glands of these animals – to "sew" to the genes responsible for the synthesis of the human proteins we need. Mammary glands belong to the glands of external secretion, which means that even the most active proteins that are produced there will not affect the animal giving milk. It is important and very difficult to create such a structure so that it does not have ectopic expression, that is, the protein is produced only in the mammary gland. "In the jargon of geneticists, this is called "making sure that the promoter does not leak," says Nariman Battulin, head of the stem cell genomics sector. So my work in this project can be compared with the work of a plumber who makes sure that it does not flow where it is not necessary, and with the work of a geologist who determines the location and influence of the surrounding landscape."

"We created five designs with different promoter lengths and tested them on mice, because it is cheaper and more accessible," explains project manager Irina Serova. – Of the five designs, only one came up (oddly enough, with the longest promoter) – the protein was released only by the mammary gland and only during lactation. We created the first designs based on the cow gene, but then it became possible to use goat genes. Thus, the regulatory region of the alpha-S1-casein gene of goat was connected with full-sized genes of granulocyte-colony-stimulating or granulocyte-macrophage colony-stimulating human factors."

The complex process of creating gene constructs is called "cooking" in the jargon of geneticists. Genetic engineer Gennady Dvoryanchikov was engaged in cooking the structure for this experiment. "Now cooking is a fairly ordinary process, even undergraduate students are engaged in it. But when we created the designs, the work required a lot of talent. We looked at genetic engineers then as gods," my interlocutors explain with a smile.

In the closet next to the table of Candidate of Biological Sciences Irina Serova is a small statuette of a goat – somewhere in Brazil there is exactly the same transgenic living original. Irina Alexandrovna puts the statuette on the table in front of me and thoughtfully runs her finger along the withers: "This is our Camila. Brazilians are, you know, sentimental people. Here, a sculptor was invited specially, they made a statuette of Camila from nature. When our goats were born, we became stars of Brazilian television, newspapers and magazines for a while. These were the first transgenic animals born in Brazil. So our Brazilian colleagues are very grateful to us – we have brought the not very well-known Ceara State University to a new level."

"Why Brazilian, and not Russian?" – I ask.

"We don't have such conditions. Their science is focused on agriculture. Of course, we had to fulfill a lot of grants to bring the laboratory to the right level, but they had a foundation and specialists."

The ICIGA laboratory was not the only one looking for partners abroad for such a project. Another well-known Russian project (led by Igor Goldman, Director of the Transgenbank Institute of Gene Biology of the Russian Academy of Sciences) with transgenic goats, from whose milk lactorferrin is extracted, was launched on Belarusian animals.

Another pride of Brazilians is that a local, Brazilian breed of goats was used. Irina Serova admits that initially they considered the option with more dairy Swiss goats.

"The injection is performed at the stage when the male and female cells have just merged. But it turned out that if the cytoplasm of mice is transparent, then goats, sheep and pigs have a lot of fat capsules floating in the cage, and we don't know where to prick. We had to select a breed that had less of this fatty substance. In addition, the eggs of the selected breed had to be resistant to soft centrifugation (thus, the fat capsules shift to one pole of the cell, opening visibility)," Irina Serova tells about the advantages of Brazilian goats.

In fact, it is at this magical moment of the birth of a new life, when maternal and paternal genes merge, geneticists "under the guise" and add new pieces of DNA. It is not yet possible to say in advance exactly where these pieces will be embedded: they can stand in some gene, they can stand between genes. Nariman Battulin compared theoretical studies of exactly where the structure stands, how genes that happen to be nearby affect it with the work of a geologist. In this case, the researchers were lucky: the created design turned out to be very stable, regardless of the embedding location and the number of embedded copies, the amount of protein was expressed approximately the same, ectopic expression was not observed.

Now Siberian scientists have proved that the granulocyte-colony stimulating factor isolated from the milk of transgenic goats and mice is identical to human. Its effect on hematopoietic cells – precursors of human umbilical cord blood - has been tested. Chinyu and Camila became the founders of two lines of transgenic goats. They have already had offspring who have successfully inherited the ability to produce such an important human protein for hematopoiesis.

"Is it possible to drink this milk?" – I can't resist a banal question. "Why? – Nariman answers the question with a puzzled question. – Although, I think it won't be a big deal. Our gastrointestinal tract digests proteins very successfully, and most of them do not enter the bloodstream." But still, Chinya and Kamila's newborn goats were fed with milk by special nurse goats. Here the question is not so much about the safety of milk, but about its price and importance for other purposes.

Portal "Eternal youth" http://vechnayamolodost.ru10.07.2014

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