Continuation of the possibility of cloning of oviparous animals

Individuals with similar plumage phenotypes appear in the offspring of "cloned" somatic cells of wolf mountain chickens. Courtesy of Yangzhou University

■ Reporter Li Chen Correspondent Yu Lu

Since the advent of dolly clone sheep in 1997, more than 20 species of mammals have successively achieved asexual reproduction of somatic cloning. However, due to the special oviparous reproduction model, somatic cloning methods cannot be used in poultry animals, so the work of bird breeding, rescue, endangered recovery and bioengineering breeding has encountered bottlenecks and has not been broken.

Recently, the journal Nature Communications published the latest research articles by Professor Li Bichun and Chen Guohong's research team in the College of Animal Science and Technology of Yangzhou University online. This article introduces the theoretical research and methodological system of reversing chicken body cells (fibroblasts) into primitive germ cells to reproduce offspring.

Crack the avian somatic cell cloning puzzle

Chen Guohong, co-corresponding author of the article, told China Science Daily that the principle of mammalian somatic cell cloning technology is to transfer reprogrammed somatic cells into denucleated oocytes, form reconfigured embryos, and then transfer them into the surrogate mother to continue to develop, resulting in cloned animals, such as cloned sheep "Dolly".

"But this technology cannot be implemented on poultry." Li Bichun, co-corresponding author of the article, said. The main reason is that "avian fertilized eggs (eggs) are excreted to the late blastocyst stage with a large number of cells and are closely linked to the yolk to absorb nutrients." ”

Team member Zuo Qisheng introduced that in addition to oocytes (unfertilized eggs) or early embryos (fertilized eggs), poultry eggs also contain a large number of nutrients needed for embryonic development (yolk); in addition, the nuclei of poultry oocytes are large and only surrounded by a thin layer of cytoplasm, which makes the process of removing and moving into the nucleus difficult to implement.

If the mammalian somatic cell nuclear transfer method is followed, the reconstructed embryo is implanted into the egg, which is equivalent to removing the embryo that has been "implanted" in the womb, and then replacing it with the reconstructed embryo to continue to develop.

"This process is currently impossible to achieve on oviparous animals." Zhang Yani, author of the article, said, "This makes the cloning of poultry animals encounter insurmountable problems, and this bottleneck hinders the research progress in the fields of poultry genetics, developmental biology and poultry bioengineering breeding, germplasm resource conservation and endangered poultry restoration, as well as genetic molecular mechanisms." ”

At present, China has very rich resources of livestock and poultry breeds. Chen Guohong believes that doing a good job in the protection of livestock and poultry breeding resources is the basis for the utilization of excellent traits and biological engineering breeding in the future, and it is particularly necessary to excavate and establish a suitable breeding system.

In mammals, both somatic cloning and gamete and embryo cryopreservation techniques can effectively protect current breed resources. But in poultry, these methods are not fully applicable, so new methods need to be pioneered.

"Our study takes advantage of the characteristics of chicken primitive germ cells migrating with blood, first of all, donor cells, that is, fibroblasts, are transdifferentiated into primitive germ cells in vitro, and then allogeneically transplanted into the receptor, so that they borrow the environment of the receptor gonads to develop into sperm or eggs, and when the recipient chickens are sexually mature, they mate with each other to produce offspring from donor chickens." Because of its differences from mammalian somatic cell cloning, we call it a clone-like chicken. Li Bichun said.

"The emergence of clone-like chickens can not only provide a new method for the protection of germplasm resources of poultry animals and the restoration of endangered birds, but also provide a new way for basic research work such as bioengineering breeding and analysis of trait genetic mechanisms of poultry animals in the future." Chen Guohong said.

Achieve efficient somatic reversal into primitive germ cells

The team launched research on clone-like chickens in 2010. With the continuous study of the biological characteristics of chicken primitive germ cells, they found that chicken primitive germ cells can be used as tool cells for the production of clone-like chickens, but encountered the problem of how to obtain a large number of primitive germ cells.

Although a highly efficient method for inducing primitive germ cells in vitro has been established in mammals, it has not been established in poultry.

Zuo Qisheng introduced that the team has established a model of primitive germ cells migrating to the nest and producing offspring chickens in the early stage, which provides a new idea for the protection and restoration of germplasm resources of chickens.

But because of the small number of primordial germ cells sourced from isolated chickens and the sacrifice of early embryos, the team has been exploring ways to obtain a large number of primitive germ cells.

They first established a system for embryonic stem cells to induce differentiation into primitive germ cells in vitro, but embryonic stem cells are also not easily available. "Somatic reprogramming technology provides a good idea for our research, somatic cells can be reprogrammed to form induced pluripotent stem cells with differentiation potential similar to embryonic stem cells, and somatic cell isolation and freezing technology is relatively easy, can be obtained in large quantities in a short period of time, and will not cause great harm to individuals." Zuo Qisheng said.

Therefore, they selected chicken fibroblasts, first reprogrammed them into induced pluripotent stem cells, and then further induced differentiation into primitive germ cells, which can greatly solve the problem that primitive germ cells cannot be obtained in large quantities and are difficult to isolate and culture.

After more than 10 years of exploration and research, the team reversed the somatic cells of China's black-feathered wolf mountain chicken into primitive germ cells, and transplanted it into the recessive white-feathered WhiteLock receptor chicken (surrogate chicken), producing offspring from the donor black-feathered wolf mountain chicken.

Somatic recovery into live birds becomes a reality

"This technology is a new method of restoring somatic cells into living organisms, an innovation that changes the traditional rules of poultry bioengineering breeding and germplasm resource conservation, and has important practical significance for the development of the poultry industry." An article review expert commented.

"This research, which uses somatic cells from chickens to produce offspring, is a game-changer for the production of cloned chickens and is very useful in the field of chicken research." Another reviewer commented.

After exploration, Li Bichun and Chen Guohong's research team also systematically analyzed the regulatory mechanism of chicken cell reprogramming and primitive germ cell generation, and established a method system of efficient somatic reprogramming and inducing pluripotent stem cells to differentiate into primitive germ cells and primitive germ cell allogeneic transplantation.

"Using a combination of these three techniques, somatic cells have been successfully restored into live poultry. This work breaks through the bottleneck of the difficult operation of poultry genetic engineering, and provides theoretical basis and technical support for the protection of poultry animals and the restoration of endangered species. In the future, combined with gene editing technology, genetically modified offspring can be effectively obtained and the bioengineering breeding process of poultry animals can be accelerated. Li Bichun introduced.

At present, the team has also established a rapid and effective technical system for cryopreservation of chicken skin tissue, and can obtain cells from thawed and recovered skin tissue and induce them to become primitive germ cells, providing technical support for the long-term preservation of poultry germplasm resources.

Source: China Science Daily