The book "The Science of Resurrection species. How to clone mammoth "

We dream to live forever. We hope that we can clone our beloved pet, as happened with Dolly the sheep. We want to walk around the “Jurassic Park”, look at dinosaurs and mammoths, see extinct moas, dodos and other creatures.

Beth Shapiro, a professor at the Department of Ecology and Evolutionary Biology at the University of Santa Cruz in California, tells us the fascinating history of modern science of recreating species.

As soon as any organism dies, its DNA immediately begins to collapse under the influence of ultraviolet radiation and bacteria, therefore one cannot simply take a cage and clone an extinct animal. Researchers have to deal with a difficult task - they are trying to put together a puzzle in which some of the pieces of DNA are lost.
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Let's find out whether we need to restore the extinct species (Beth Shapiro is sure that it is worth it), what difficulties await us on this path and what it can lead to.

Excerpt
CREATE A CLONE

When you work in the tundra, no one cares that you are falsely singing at the top of your voice, walking along a meandering river. No one laughs at the five layers of clothes worn on you, and does not make fun of the variety of nets that you entangled yourself in the last doomed attempt to keep mosquitoes away from your body. No one leads by the ear when your battered MI-8 helicopter makes an unexpected landing in the middle of Siberian tundra in order to pick up a French-speaking couple with a five-year-old child and a big red fridge.

I learned all this in the summer of 2008, during what I fondly recall as my strangest and least successful bone hunt season. That summer we spent several weeks in a small camp surrounded by lakes in the lowland tundra of the Taimyr Peninsula. We hunted mammoths.

Bernard Byuig, an experienced and eccentric Arctic explorer in a good way, led the expedition to Taimyr, and there was no reason to believe that we would fail. For decades, Bernard led the company Tserpolex (from the French CERcles POLaires EXpédition) and led land expeditions in Siberia and the North Pole. These expeditions began at its well-equipped base in Khatanga, a small Russian town, standing on the Khatanga River in the Krasnoyarsk Territory. By the beginning of the 2000s, Bernard switched to expeditions of a more scientific nature and founded the organization Mammuthus (Latin mammoth) under "Tierpolex", whose stated goal was to research and glorify the Arctic and its many treasures. However, as the name of this organization hints, the focus of her particular attention was the search for the mummified remains of mammoths and the promotion of their research. The formation of the Mammuthus company was either an adventurous step, or simply very timely, since from the beginning of this century the mummies of mammoths and other ancient ice-field giants began to be discovered in the permafrost of Siberia, surprisingly often.

Having met with Bernard, it was impossible not to be sure both in his leadership qualities and in the success of the expedition. By 2008, Bernard had dozens of years of experience in the Siberian tundra. He possessed inexhaustible energy and enthusiasm, was well acquainted with the difficulties of logistics in working in Siberia (and knew ways to circumvent these difficulties), and also owned a large collection of warm jackets. Most importantly, he worked with the local population for a long time, and this explains in some way why he was so often the first to get access to the newly discovered mammoth mummies. Everything indicated that the expedition should be crowned with success.

Our adventure began in the Siberian home of Bernard in Khatanga. Khatanga is an unusual place. This is one of the most northerly points in the world where people live. Although the population of the city is less than 3.5 thousand people, there is an airport, a hotel and a museum of nature and ethnography, full of exhibits related to people living in this area and its history. Khatanga also has several restaurants serving local meat, seasoned with dill, and several small shops selling carrots with signs of frostbite for $ 8, semi-automatic machine guns and a fancy variety of flavored chewing gum. Roads and river banks are littered with unfamiliar mechanisms, some of which may still be working. People live there anywhere - and in small wooden huts, and in large apartment buildings and even transport containers - those used on container ships for the transport of goods across the ocean. Even the house of Bernard consisted partly of shipping containers, joined together and supposedly well isolated from the external environment. After all, the city is located at 71 degrees north latitude, and winters in Khatanga are dark and cold, with an average monthly minimum temperature of around –35 ° C and no sunlight at all for many days in December and January. True, we stayed there from July to August, and the air temperature fluctuated within acceptable limits of 5–15 ° C, and the sun shone around the clock. Of course, several mosquitoes were circling around, spoiling the rest of the wonderful atmosphere. More precisely, a few hundred mosquitoes.

Per cubic centimeter of air.

Our expedition included Bernard, his wife Sylvia and their twenty-year-old nephew Pete, several Russians who worked for Bernard, a French woman director and her boyfriend, as well as an entire gathering of scientists with a wide variety of interests concerning ice-age animals. The most senior scientist in our group was Dan Fisher, a mammoth specialist and professor at the University of Michigan. Dan is a global expert in his field: exploring the growth patterns of mammoth tusks, he can determine the gender, reproductive history, lifestyle, and even the cause of death of the animal. Dan also measures the amount of stable isotopes of chemical elements, carbon and nitrogen, accumulated in the mammoth's tusk as it grows. These isotopes form an almost continuous record of changes in the diet of the mammoth and in its environment. Adam Rowntree and David Fox, previously trained under Dan, also worked with us. Finally, there were two researchers among us interested in DNA: I and Ian Barnes, who at that time taught at Royal-Holloway College at the University of London, but I met him when I was working on my dissertation at Oxford University.

Dan, David, and Adam dreamed of finding tusks, but Ian and I hoped for the mammoth bones. Tusks are better suited for isotopic analysis, but they contain very little DNA. Ian and I were also interested in all the animals that lived on Taimyr during the periods of glaciation, so that we were not strictly focused on collecting mammoth bones.

For reasons that remain a mystery to me, and despite promises made to Bernard even before our arrival in Khatanga, we had to wait a whole week for the helicopter. We temporarily settled with Bernard and, to kill time, took up the exploration of Khatanga. We tried on a lot of warm jackets and mosquito devices. We roamed the streets, teasing local dogs and trying to unravel the purpose of various mechanisms. We set traps for insects and determined the types of those that got there. We have drilled holes in several bones from the Bernard collection for our film crew and for the benefit of future research projects. While we were waiting, Bernard organized and was involved in one after another meeting with his group of Russian scientists and logistics specialists. These meetings were bright and exciting: giant cards did not fit on the tables, conversations proceeded to an elevated tone, reconciliations were carried out with old scientific documents describing the geographical limits of past glaciations, vodka was poured into glasses and a plan was made for a future excursion.

Finally, the helicopter arrived and it was time to fly into the field. We collected food, fuel, and belongings and left the house of Bernard for the airport. We made our way through security control to the runway and came face to face with our next vehicle: the all-favorite Mi-8 helicopter. About a quarter of the space in it already occupied two huge gas cylinders. Going past the cylinders, we threw inside our camping equipment, cameras and lighting devices for filming, two large inflatable boats and two outboard engines with a capacity of 250 horsepower each, enough rice and unknown freeze-dried food to feed twenty people for six weeks. , a giant canister of gasoline for cooking and vodka in an amount sufficient to feel happy for at least a day. About a third of the windows lacked in the Mi-8 helicopter, presumably to make smoking more comfortable on board.

Having loaded all our belongings, we climbed inside and settled on the benches under the windows, as well as on top of things and gas cylinders. Pasha, our cook's dog, a one-year-old Siberian Husky, was the last to board. Pasha expressed his concerns about participating in our expedition, trying to merge with the covering of the runway under the ladder. I shared Pasha's doubts as to whether it was better to be swallowed by the runway or to go up in the sky on the Mi-8. When it became clear that the strip did not want to absorb Pasha, he escaped. The cook and one of the pilots climbed out, smoked a few cigarettes, caught Pasha, lifted him up to about the middle of the ramp, somehow managed to miss him, caught him again, pacified enough to drag him to the end of the ramp and put him in the door, and finally we settled in the cabin. Under the cheers and desperate howl of Pasha, we left the ground and flew toward the tundra.

Somatic nuclear transfer

If so many bones have already been accumulated in collections all over the world, why should we get out in the field to find some more? Why deal with broken helicopters, gold mines, twenty-four hours of daylight and clouds of mosquitoes? The answer is simple: the best bones are those that came to us straight from the icy tundra. We want to find bones that have never thawed. They contain the best preserved cells with the best preserved DNA.

We are not the only group of scientists who spend their summer in the Arctic in search of the remains of animals of the Ice Age or dangling in gold mines, but I am pleased to think that we have the most sensible approach to business. For example, we know that we are not looking for cells that can be cloned. All that scientists know about cloning animals using somatic cells (that is, not sperm cells or eggs), says that cloning will work only if the cell contains an intact genome. No such cells were found in the remains of extinct animals found in the ice of the tundra.

DNA destruction begins immediately after the death of the organism. Plant and animal cells contain enzymes whose task is to sever bonds within the DNA molecule. These enzymes, called nucleases, are found in cells, tear fluid, saliva, sweat, and even on the tips of our fingers. While we live, nucleases are critically important to us. They destroy pathogenic microbes that enter our bodies before they cause us any harm. They eliminate damaged DNA, allowing our cells to repair what was broken. And after the death of our cells, nucleases destroy their DNA, so it’s easier for our bodies to get rid of them. In other words, nucleases have evolved in such a way as to remain active even after the cell dies, and this is bad news for those who want to clone a mammoth.

In the laboratory, we do not allow nucleases to destroy the DNA that we are trying to isolate, either by immersing a fresh sample in a solution of chemical inhibitors, or by subjecting it to rapid freezing. The Arctic is a cold place, but not cold enough to freeze something (especially as big as a mammoth) fast enough to protect DNA from decay. In addition, nucleases are produced by all living organisms, including bacteria and fungi, which colonize decaying bodies of dead animals. Consequently, the chance that the genomes of any cells can remain completely intact for a long time after death is small. Without an intact genome to clone a mammoth will not work. More precisely, it will not be possible to clone a mammoth by somatic nuclear transfer.

Somatic nuclear transfer is a sad, but quite appropriate name for the process, thanks to which we have, in particular, the most famous clone - Dolly the sheep (Fig. 8). Dolly was cloned by scientists from the Roslinsky Institute in Scotland in 1996. Scientists removed the nucleus — the part of the cell containing the genome from the mammary gland taken from an adult sheep — and placed this nucleus in a prepared egg from another adult sheep. Then this egg developed in the womb of another adult female into a completely healthy individual of its own species. It is important to note that a sheep cloned by nuclear transfer was genetically identical to an animal that had donated a mammary gland cell, and had nothing to do with its surrogate mother or that sheep from which an egg cell was taken.


To understand the intricacies of this process, you need to learn something about the cells. Our bodies (and the bodies of other living organisms) consist of cells of three main types: stem, sex, and somatic. Somatic - most of all, these include skin cells, muscle cells, heart cells, etc. Somatic cells have a diploid set of chromosomes, which means that they contain two copies of each chromosome - one from the mother and one from the father. Somatic cells also have a specialization — these could be brain cells, blood cells, or breast cells, like those used to create Dolly. Another category of cells is the primary germ cells (gonocytes), from which gametes are formed - spermatozoa and egg cells. Gametes have a haploid set of chromosomes, that is, they contain only one copy of each chromosome. In normal sexual reproduction, two haploid gametes merge at the time of fertilization, forming a diploid zygote, from which the embryo then develops.

During nuclear transfer, the stage of fertilization and fusion of gametes is omitted. Instead, a process called enucleation occurs, during which the haploid ovum genome is removed. Then a diploid nucleus of the somatic cell is placed in its place (in the case of Dolly, the mammary gland cells).

In normal sexual reproduction of mammals, the zygote formed during fertilization contains cells that have no specialization. Such unspecialized cells belong to the third category and are called stem cells. The stem cells that make up the zygote at an early stage of their development are called totipotent, because they can turn into cells of any type and, therefore, can give rise to a whole living organism. As the embryo develops, the cells multiply and begin to differentiate, that is, perform more specialized functions in the body. At one of the earliest stages of embryo development, totipotent stem cells lose their ability to transform into cells of any type, but still do not have a clear specialization. Now these cells are called pluripotent. Mammalian pluripotent stem cells, for example, can transform into cells of any type other than placental.

Pluripotent stem cells are of particular interest to science because they can be used to treat people. When stem cells divide, either stem cells or specialized somatic cells are obtained from them. This means that they are potentially capable of replacing diseased or damaged cells. Stem cells can be found not only in the developing embryo, but also in all tissues of an adult organism. Adult stem cells are prone to higher specialization than fetal ones, but despite this, they are crucial for the repair of damaged tissues and their renewal. For medical purposes, adult stem cells are often taken. For example, hematopoietic stem cells can turn into different types of blood cells, and they are used in the treatment of blood diseases, including leukemia.

Let's go back to nuclear transfer cloning. Somatic cells, unlike stem cells, are highly specialized. They cannot transform into different cell types, since they represent the end point of the differentiation process. Somatic cells have a specific function, and their cellular mechanisms are adapted to the quality performance of this work. In a somatic cell taken from the mammary gland of a sheep, only those proteins that are necessary for it to perform the function of the mammary gland cells are expressed, and therefore only those genes that encode these proteins are included in it.

In order for a somatic cell to become a whole living organism, it must “forget” everything about its specialization and dedifferentiate. It must again become an embryonic stem cell.

Although Dolly is perhaps the most famous animal born by somatic nuclear transfer, she was not the first clone created in this way. In the 50s and 60s of the twentieth century, John Gurdon of Oxford University proved that frog eggs develop into frogs even after the nuclei of these cells were seized and replaced by somatic cell nuclei. Although at that time the mechanism of this phenomenon was not well understood, the key observation of Herdon was that the egg somehow starts the process of dedifferentiating the somatic cell - and the latter “forgets” what type of cell it was before. In 2012, Gurdon received the Nobel Prize for this discovery, along with Shinya Yamanaka of Kyoto University. Yamanaka later discovered that the same pluripotency (dedifferentiation of somatic cells) can be achieved in vitro, that is, in tissue culture under laboratory conditions, by adding a set of transcription factors into the cell, which are proteins that combine with certain sections of DNA and control which genes should turn on and when. These cells are called induced pluripotent stem cells (iPSC).

Nuclear transfer is used to clone sheep, cows, goats, deer, cats, dogs, frogs, ferrets, horses, rabbits, pigs and many other animals. Also gaining popularity is the cloning of animals with specific desired properties. The Internet is widely advertised commercial services involved in the cloning of pets and the creation of cloned offspring of horses-champions. The first results are already visible: at the end of 2013, Shaw Mie's six-year-old horse, a clone of the Sage mare that competed in horseback polo, became the champion of the Triple Crown in Argentina, possibly heralding a new era in animal breeding for shows and sports.

However, cloning by nuclear transfer is of low efficiency. Dolly was the only embryo out of 277 created at the Roslinsky Institute, which lived to its birth. A mare named Prometheus, the first cloned horse to be born, was the only embryo out of 841 that developed into a full-fledged individual of its own species. Snoopy, an Afghan hound dog, cloned by Korean scientist Hwang Wu Sok, became one of two puppies born after 1095 embryos were implanted with 123 different surrogate mothers, and the only one who lived for more than a few weeks. In all these cases, scientists had access to a potentially infinite number of somatic cells taken from living animals.

Living mammoths do not exist.

»More information about the book can be found on the publisher's website.

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