Release date: 2017-12-28
In the pig farm researched by the Lai Liangxue team of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, a group of special pigs lived. There are not only a dozen tool pigs with genetic scissors, but also six or seven hundred genetically modified pigs that have important application value in the biomedical field. The use of these pigs sounds more "stunned". Some genetically modified pigs can mimic human diseases such as Alzheimer's disease, gradual freezing of human disease and Huntington's disease. Some pigs can provide a source of xenogeneic organs for human organ transplantation.
Xenotransplantation has always been one of the key directions of international research. If this major difficulty can be overcome, it will bring hope to millions of patients around the world who need organ transplants.
What's exciting is that this door to hope is quietly opening up. Not long ago, Lai Liangxue's research group published a new model pig model for conditional expression of Cas9 gene in the international authoritative journal Genomics. paper.
For the first time, the Guangzhou Institute of Biological Sciences has developed a tool with genetic scissors.
Why choose pig
To understand this research, start with the Cas9 gene.
Cas9 gene and CRISPR technology have been used in the Zika virus outbreak, used to monitor Zika virus, and even used to detect SARS, SARS coronavirus, measles virus, influenza virus, hepatitis C virus. The former is a nuclease in the gene, and the latter is the abbreviation of "regular clustering interval short palindrome repetition", from the body of bacteria, shouldering the "heavy duty" of bacterial immunity.
Cas9 is a key nuclease in the process of CRISPRing the virus by self-transformation of viral DNA. But this time, the two together did not want to detect the virus, but the researchers buried in the pig a "secret" that can be inherited from generation to generation, let it shoulder a larger mission.
The secret is that researchers have modified the pig's genome by a fixed point to open a "convenience door" for the experiment.
The advancement of medical technology has always been inseparable from the sacrifice of experimental animals. This is because in order to obtain new knowledge about biology, medicine, etc. or to solve specific problems, human beings cannot experiment on themselves, and only use animals in the laboratory for scientific research.
Among the animals used for the experiment, there are small animals such as mice, rats, rabbits, and large animals such as pigs, orangutans, and baboons. However, because the gestation period, growth period and sexual maturity period of large animals are longer, it is not as short as the breeding time of small animals such as mice, rats and rabbits. "If you do genetic experiments, you need to change the genes during the embryos, and then put them into the surrogate mother, waiting for the birth." Lai Liangxue told the Journal of the Chinese Journal of Science and Technology, "The mice are 21 days pregnant, 4 weeks can be sexually mature, continue Breeding the next generation. But pigs have about 114 days of pregnancy and 6 to 8 months of sexual maturity. So researchers often choose small animals with shorter incubation periods to see the results soon.
However, although the use of small animals has shortened the time, because the difference between small animals and human body is large, the experimental results are often not directly used in humans. So who can shoulder the heavy responsibilities, be close to humans, and make the experiment closer to reality?
In many mammals, although the pig is not close to human blood, its organ size, morphological structure, physiological metabolism and immune system are very close to human beings. Moreover, pigs are human-raised livestock, and the number is higher than that of baboons, orangutans, etc. There are more primates and no ethical obstacles, so pigs are currently the ideal experimental animals.
But the problem is that if you want to knock out a specific gene according to the purpose of the experiment, the researcher must start from the embryonic stage of the animal, and the experiment can be carried out until the animal is bred. This not only prolongs the experiment time, but also because the cultivation process is complicated and the success rate is relatively low, resulting in a large overhead.
Lai Liangxue, who has been engaged in genetically modified cloned pig research for nearly 20 years, realizes that if a pig that can directly edit genes in the body is cultivated, the above problem can be solved.
2018 (2nd) Model Workshop on Animal Models and Major Diseases Research and Application
Dr. Wang Kepin, Ph.D., the first author of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, explained in an interview with the Journal of the Chinese Academy of Sciences: "We envisage that we can add protein genes capable of cutting the genome to the genome of pigs through gene editing technology. This is equivalent to inserting a genetic scissors into the pig's body. Moreover, the first generation of tool pigs with gene scissors obtained by somatic cell cloning can also be genetically inherited by simple breeding to form genes with genes. The pig population of scissors. This allows the experimenter to experiment directly on adult pigs without having to start from the embryonic stage, simplifying and greatly speeding up the experimental process.
Four years of persistence
After finding the research direction, in 2013, Lai Liangxue led the team to skillfully design the experimental plan, and the next task was to keep moving toward the goal.
During the experiment, the researchers used gene targeting technology to insert the Cas9 protein gene that can cut the gene into the specific locus ROSA26 of the pig genome. "Cas9 protein is used as a scissors and is embedded in the pig gene in advance. However, in order to prevent Cas9 from being cut, a switch has been added," Lai Liangxue explained.
The key to controlling this switch is the Cre recombinase. Cre (Cyclization Recombination Enzyme) is an enzyme protein derived from phage P1, which recognizes homologous recombination between two LoxP sites in a catalytic gene, resulting in DNA deletion, translocation, etc. phenomenon. This property has been widely used in genetic engineering operations, especially in the establishment of inducible knockout mice.
So, can this technology be applied to pigs to be successful? “We did it very well, because we designed the experiment better, so the first generation of pigs got the desired results when they were born.†Lai Liangxue recalled.
In fact, the reason why the experiment was so smooth, but also benefited from the "cultivation" of Lai Liangxue for many years. Since returning to China in 2007, he has been engaged in the construction of genetically modified pig models. Previously, he has made major breakthroughs in various genetically modified pigs. A good foundation has ensured the smooth progress of this tool.
Wang Kepin is a graduate student of Lai Liangxue's research group. He also clearly remembers that one day in April 2015, the cloned tool pig was born. “The pigs are born two or three in the afternoon. We need to use molecular experiments to verify whether the pigs are the tools we need.†In order to get the results as soon as possible, Wang Kepin and other researchers together in the laboratory for identification.
That night is doomed to sleep. Wang Kepin and his colleagues have been busy from the afternoon until 9 am the next morning. When the results of the experiment showed positive, they were particularly excited - they succeeded!
Whether it is the first generation or the descendant tool pigs that have been bred, the appearance and other functions are no different from normal domestic pigs. "They don't have specific diseases." Lai Liang said.
The key point is that if the boar and the sow are genetically modified at the time of breeding, not all of the offspring will be positive; if both the boar and the sow have completed the genetic modification, then all the offspring will be 100% Inherited this gene.
This means that when experimenting with a tool pig, the experimenter does not need to modify the pig's genome every time. It is only necessary to insert the Cas9 protein gene at the genome-specific site of the first generation, F0 generation pig, and you can rely on the pig. The natural reproduction yields the same type of pig that is born with a genetic scissors, that is, a tool pig.
This is just the beginning
Lai Liangxue also used this technique on dogs while waiting for the breeding results of the tool pigs. In 2015, Lai Liangxue led another team of researchers to use the same method in the embryonic period of the dog, which knocked out some of the genes. This allows the puppies that are bred to exhibit more muscular and athletic abilities.
Because dogs are very similar to humans in terms of nutritional metabolism, physiological anatomy, cardiovascular system, etc., and dogs are smarter, they are used for behavioral research. It is suitable for establishing dog models such as Parkinson's syndrome and Alzheimer's disease to study humans. disease. Of course, dogs as pets can be used for excellent genetic traits by genetic knockout.
After the tool pigs were bred, the researchers of the Lai Liangxue team began to study the potential application value. The first thing they thought of was the use of genetic scissors to make a primary cancer model.
Most of the tumor models previously prepared for tumor pathogenesis research and drug development are achieved by transplanting human tumor cells into nude mice. "Nude mice are completely knocking out all the immune-related genes in the embryonic period, that is, they are born without any immunity." Lai Liangxue further explained, "The mouse tumor model prepared in this way, On the one hand, because there is no immunity, on the other hand, it is not a primary tumor, so it is very different from the occurrence and development of human tumors. More than 95% of the results of its effectiveness and safety test for tumor drugs cannot be converted into Clinical application."
The researchers packaged gRNAs containing Cre recombinase and gRNAs that target six tumor-associated genes to infect the lungs of the tool pigs by nasal infusion, which causes cancerous mutations in the genome of pig lung cells. Sure enough, three months later, the tool pig showed typical lung cancer symptoms and pathological changes, thus successfully establishing a large animal model of primary lung tumor.
"This tool pig can play an important role in cancer research because cancer itself is a genetic mutation," Lai Liangxue explained. As a result, other cancers in the human body can also be replicated on tool pigs in a similar manner.
“In the past, a large animal experiment cost millions of dollars. Now with the tool pig, it not only shortens the time, but also reduces the cost to tens of thousands of yuan.†Lai Liangxue said.
Make allogeneic transplantation closer to the clinic
Undoubtedly, the research of the Lai Liangxue team has made the transplantation of xenotransplants a big step forward.
It is well known that xenotransplantation is one of the key directions of international research. In 2015, Chinese and American researchers at Harvard University published a report on the online edition of Science in the United States. They used a new gene editing technology to eliminate potentially harmful viral genes in the pig genome and to conquer pig organs. A major difficulty in human transplants brings hope to patients who need organ transplants.
This is because there are approximately 11% of the repetitive elements (PRE-1) in the pig genome, which is almost identical to the copy of the repetitive elements of the primate. Research by researchers in the journal Nature reported that the structure and function of these pig repeats are very similar to replicas of primate repeats, suggesting that there is a closer relationship between humans and pigs than previously thought. many.
Now, Lai Liangxue uses CRISPR/Cas9 technology to transform the pig's genes and obtain a variety of genetically modified pigs with more immunogenicity and human body, which greatly reduces the rejection of human xenogeneic organs and makes the allografts more clinical. The closer it is.
In addition, they are exploring the possibility of using genetically engineered pigs and pluripotent stem cell technology to develop human organs. “For example, the development of the pancreas is controlled by a key gene, the PDX1 gene, so we only need to knock out this gene. Let the pigs not grow their own pancreas. In the embryonic stage, the human pluripotent stem cells are injected into the early developmental embryos. In theory, the pig's pancreas is all derived from human cells, then the pig grows up. The pancreas can be matched with humans," said Lai Liangxue.
This is not a hole in the wind. In 2016, the research team at the University of California, Davis, was trying to cultivate human stem cells by injecting human stem cells into pig embryos, and hoped that these "chimera" embryos could Provide answers to alleviate the shortage of transplant organs in the world.
Tool pigs, pigs that cultivate human organs... These are the research contents of the Lai Liangxue team. He has continuously added the understanding of CRISPR/Cas9 technology. “Now my students are already blue, and their discussions can always keep up with the world's technological frontiers.†This makes Lai Liangxue more excited.
In 2016, Wang Kepin, who completed his doctoral thesis, chose to stay in the team of Lai Liangxue. He hopes that in the future, he can make more contributions to life sciences and medicine by using large animal genetic modification models.
Source: Chinese Journal of Science
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