How did China cloned sheep come into being?

On April 16, Huada Gene Research Institute announced to the media that the "Major Project of Breeding New Varieties of Transgenic Sheep of the Ministry of Agriculture" jointly carried out by the Institute, Shenzhen Huada Ark, Institute of Genetics and Developmental Biology of Chinese Academy of Sciences and School of Life Sciences of Shihezi University made a breakthrough, and the first hand-made transgenic cloned sheep was successfully born.

Pumbaa, with short stature and yellow fur, has gathered two efforts of China researchers: the technology of artificial cloning reduces the cost and difficulty of cloning, which is conducive to further popularization; Transgenic sheep can not only produce meat, milk and wool, but also synthesize unsaturated fatty acid Omega-3 rich in deep-sea fish oil.

If all goes well, Pumbaa itself will become healthier because of this special gene; In the future, it is possible for human beings to eat mutton instead of expensive deep-sea fish oil.

"This is a research that kills two birds with one stone," said Runlin Ma, a researcher at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

Cloning technology that high school students can do.

Before Pumbaa was born, it had some friends with similar "life experiences". Professor China from Harvard Medical School got transgenic mice and transgenic pigs in 2004 and 2006 respectively. In 2008, China Academy of Agricultural Sciences made transgenic cloned pigs, and two years later, a professor in Inner Mongolia also made transgenic cloned cattle.

This time, the researchers turned their attention to sheep.

In order to choose a suitable clone, the researchers went to a sheep farm in Xinjiang. There, according to the performance and daily records of each sheep in the sheep farm, they selected a ram with good meat quality and much hair. The staff took cells from the edge of the ear as "seeds" for cloning. Zhang Peng said that this is because the cells on the edge of the ear are easy to collect and "do less harm to the sheep".

In the normal breeding process, the birth of a sheep should require the efforts of both parents. The sperm and eggs they provide carry half the chromosomes of both parents, and the genomes fuse during fertilization. In this way, when the lamb is born, it will be "like a father in some places and a mother in some places".

The cloned lamb Pumbaa has no father and mother in the traditional sense. Its production process is like making a brand-new "egg": researchers first prepare an egg and take out its nucleus carrying genetic information like "taking out the yolk of the egg". Then, the "yolk" of sheep somatic cells, that is, the nucleus, is stuffed back into the eggshell of oocytes. Although this brand-new "egg" comes from two different individuals, all its genetic genes come from the same sheep.

1996, British scientists cultivated Dolly, the world's first cloned sheep, in this way. For a long time after that, "cloning" was a very complicated technology. The source of difficulty is the "eggshell" outside the cell.

Outside the oocyte, there is a tough zona pellucida. This gelatinous "eggshell" wraps the oocyte to protect it from damage during fertilization and development. However, in the process of cloning, "eggshell" greatly increased the difficulty of scientific research personnel. Researchers need to fix the oocyte first, then insert a fine needle into the "eggshell" of the oocyte, extract the nucleus inside, and then insert the nucleus of the donor cell back through the same hole.

In order to complete this series of fine operations, researchers need expensive micromanipulators to enlarge egg cells by more than 100 times, and also need long-term training and practice. However, the size of egg cells is really "mini". In the process of penetrating the "eggshell", as long as you are not careful, the whole cell will be pierced.

In 2003, Du Yu Tao, who was studying for a doctorate at Aarhus University, was suddenly invited to participate in a research on cloned pigs in the laboratory. She was very surprised.

"I know nothing about cloning." She said to her mentor at that time.

What the laboratory is trying is a brand-new cloning technology. Researchers are no longer committed to accurately penetrating the "eggshell", but have changed their thinking angle: How about removing the eggshell?

After repeated experiments, Du and her colleagues used a special enzyme to dissolve this troublesome "eggshell". A solid "egg" has only a soft cytoplasm and nucleus. If you want to take the "yolk", you don't need a precise needle, just a blade.

Nowadays, in this technology called "manual cloning", the expensive micromanipulator is replaced by an ordinary microscope with a price of only110. Under the lens of 40~50 times magnification, researchers can easily find the area where the nucleus is located. As long as you use a small blade, everything will be fine, and the work of "removing the core" will be completed.

The next job is to put another "egg yolk" in it. The researchers put the nucleus of the donor cell and the "egg white" of the oocyte together and glued them together with bio-glue. Then use electric shock to make them really blend together.

"If this technology can be applied maturely, cloning will become a very simple matter." Du Dui said: In 2007, after returning from Denmark and joining the BGI, Du brought this technology back to China. In the past few years, she and her colleagues have successfully cloned nearly 100 pigs and cattle in this way. In fact, in the laboratory of Huada University, some high school students who have participated in the summer camp can try to operate after a short training.

Eating meat has the effect of eating fish.

Of course, the birth of Pumbaa is still not a simple matter. The most direct problem is that sheep are in estrus only in spring and autumn, so only in these two seasons can researchers seize the time to obtain high-quality oocytes.

20 10 10 Dr. Zhang Peng from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences came to the laboratory in Xinjiang for the first time. There is no filtration system and no air monitoring in this temporary partition laboratory. They put away the equipment, leaving only three or four square meters of activity space.

What's more, although 10 Beijing is still crisp in autumn, in Xinjiang, the weather is already very cold and the sheep are no longer in heat. Zhang Peng and his colleagues struggled there for two months, but failed to extract suitable oocytes.

"No way, we can only cross the river by feeling the stones." Zhang Peng said.

The following spring, they came to Xinjiang again. This time, the weather turned warmer and they finally found a suitable oocyte. When sheep somatic cells were introduced into oocytes, researchers also made a new attempt: "collage" a gene in the nucleus, hoping that Pumbaa could synthesize an unsaturated fatty acid-ω-3.

ω-3 is an essential fatty acid for human body. Scientific experiments have confirmed that if the intake of omega -3 is insufficient, the incidence of heart disease, diabetes, arthritis, rheumatoid disease and some cancers will increase. For a long time, humans have been using deep-sea fish oil and other foods to supplement their omega-3. This time, researchers in China hope to find another way for other organisms to synthesize this scarce ingredient.

Zhang Peng said that although deep-sea fish oil is rich in omega -3, fish can't synthesize it. They just ate a lot of algae containing Omega-3. In fact, higher animals cannot synthesize omega-3 by themselves-this gene has been lost in the process of evolution.

Now, researchers hope that through genetic engineering, sheep can regain this function. The researchers obtained the gene fragment for synthesizing Omega-3 by cloning nematodes of lower animals and transformed it into expression suitable for mammals. Then, transfer them into the genome of sheep.

Runlin Ma described his work as a "tailor": "We use tool enzymes to cut genes into fragments, then recombine them and insert them into the target genome."

In fact, this is not the first attempt by China scientists in this field. As early as 2008, researchers began to try to make cloned pigs carry the gene for synthesizing Omega-3, and it was successful at 20 10. Today, these piglets still live in pig farms in Huidong, Guangdong. The researchers confirmed that compared with ordinary pigs, the fatty acid content in their meat changed, with higher ω-3 content and lower ω-6 content harmful to health.

Runlin Ma hopes that this technology can make people no longer rely on expensive deep-sea fish oil, but can supplement the scarce omega -3 only by eating meat and drinking milk. "This can reduce the damage to deep-sea fish and let genetic resources play a role." He said.

It will take a long time for genetically modified animals to enter the market.

After the cloned embryos were synthesized, Zhang Peng and his colleagues observed them in the laboratory every day, hoping to find embryos that developed into blastocysts, because cloned embryos could only develop into a complete life after blastocysts were formed. However, for a long time, Zhang Peng was disappointed every time. Later, he even gave up hope.

Summer has come, and changes have finally appeared. Zhang Peng suddenly found a blastocyst in the laboratory. He excitedly called immediately and reported the result to his colleagues.

"We are very happy." Zhang Peng said, "This is a symbolic success: individual cells have the ability to develop into individuals, which is a process from scratch."

Five months after pregnancy, Zhang Peng and his colleagues came to Xinjiang again when Pumbaa was due to give birth. However, the production process was not as smooth as expected, because it was too late to produce, and the researchers finally gave the "surrogate" ewe a caesarean section.

Pumbaa was already dead when she was taken out of the ewe's stomach. The "nervous and scared" staff patted it and wiped the amniotic fluid from its nose. After a few minutes, it moved slowly.

Today, Pumbaa continues to grow in an experimental sheep farm. Zhang Peng said that whether Pumbaa can synthesize omega -3 by itself needs further testing, but it does carry the transferred gene.

"It is 100% transgenic animal," Zhang Peng said.

In fact, Pumbaa is not only a product of scientific research, but also a part of China's "strategic reserve project" for agricultural development. The national agricultural department has invested funds to support the cultivation of new animal varieties and the strategic reserve of biotechnology. As the person in charge of this new animal breed cultivation project, Runlin Ma said that the development of modern biotechnology has two application directions, one is to help solve human food products, and the other is to help solve people's health. The transgenic cloned sheep Pumbaa has two purposes at the same time: on the one hand, it can improve the quality and output of livestock products, on the other hand, it can improve people's health and resistance.

However, this sheep can't avoid the debate about its "origin". Both cloning and transgene bypass the normal reproductive process, and their safety has been questioned by the public.

"We understand this controversy." Runlin Ma said, "Genetically modified life can serve our specific human goals, but it will not replace natural processes. We don't have many opportunities to overcome nature, because natural selection is perfect. "

Now in China, the research and development of cloning transgenic animals are still going on. Du introduced that they had used transgenic methods to make the milk secreted by cattle and sheep also contain serum protein and milk body protein of human milk. In this way, the mammary glands of cattle and sheep are like a "small workshop" for synthesizing protein. As long as they are still secreting milk, they will continue to produce this kind of protein.

The bigger application is in cloned pigs. The researchers transferred specific genes to cloned pigs, making them more susceptible to diabetes. In this way, pharmaceutical companies can conduct drug trials more conveniently and promote the research and development of drugs for treating diabetes.

"This is the value of artificial cloning." Du said:

As for Pumbaa, who is about to have a full moon, the researchers need further tests. On the one hand, we must strictly test whether our own Omega-3 and other substances are safe. On the other hand, we should also check whether the new gene will make it have any bad changes, such as making it easier to get sick.

"What we eat will be digested into various nutrients such as fat, protein and sugar, and will not be expressed as genes in the body." Runlin Ma said, "If the substance itself is not toxic, it is safe for people to eat it."

However, at present, these technologies are still quite far from the lives of ordinary people. Du believes that the ability of the laboratory is not enough to undertake large-scale industrial production. From the perspective of safety, Runlin Ma and Zhang Peng believe that it will take a long time to observe that foods made from genetically modified animals have no side effects and negative effects.

"I personally think that it will take a long time for genetically modified animals to enter the market." Zhang Peng said.