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Breakthrough: Chinese researchers cloned monkeys using Dolly’s cloning method

The Chinese Academy of Sciences announced that the world's first monkey cloned by somatic cell nuclear transfer, "Zhong Zhong", was born on 27 November 2017 and the second cloned monkey "Hua Hua" was born 10 days later. The research results were published online in the internationally acclaimed journal Cell on 24 January 2018.

This is the first time that non-human primates are successfully cloned by somatic cell nuclear transfer (SCNT), the technology used to clone Dolly the sheep, the world’s first cloned mammal. Since Dolly’s birth in 1996, scientists from all over the world have cloned mammals such as cattle, pigs, mice, rats, cats and dogs using somatic cell nuclear transfer, but none has surpassed the barrier of cloning non-human primates.

The somatic (body) cells used in the cloning of the macaques came from an aborted female macaque fetus. The nucleus of the somatic cell is transferred to a donated egg cell, which has its DNA removed. The reconstructed cell is induced to grow into an embryo, which is then implanted into a surrogate female macaque.

DNA fingerprinting showed that the two macaques born are genetically-identical and the genes are the same as those of the donor somatic cells, proving that they are successful clones. Both monkeys were born in the labs of the Institute of Neuroscience, Chinese Academy of Sciences, in Shanghai. Researchers named the newborns Zhong Zhong and Hua Hua after the Chinese phrase "Zhˉonghuá," which means Chinese nation or people.

Zhong Zhong and Hua Hua are not the first primate clones—the title goes to Tetra, a rhesus monkey created in 1999 by a simpler method called embryo splitting (Science, v. 287, no. 5451, pp. 317-319). This approach is similar to the process that creates twins, but it can only generate up to 4 offspring at a time. Zhong Zhong and Hua Hua are the product of somatic cell nuclear transfer (SCNT), the technique used to create Dolly the sheep over 20 years ago.

According to the team leader Dr. Sun Qiang, there are three main difficulties when it comes to cloning monkeys. One of the problems is that it is difficult to extract the nucleus from the somatic cell.

The second problem is that oocytes are easily activated in advance. In the cloning process, after transferring the somatic cell nuclei into the egg cells, the egg cells need to be "waken up" to start a series of developmental program. The timing needs to be very precise. However, using traditional methods, the monkey's egg cells are often activated early, resulting in cloning programs that do not function properly.

The third problem is that the development of cloned embryos is inefficient. The vast majority of cloned embryos do not last the whole gestation stage.

Sun and his colleagues overcame the challenges primarily by introducing epigenetic modulators after the nuclear transfer that switch on or off the genes that are inhibiting the embryo development. The researchers found their success rate increased by transferring nuclei taken from fetal differentiated cells, such as fibroblasts, a cell type in the connective tissue. Zhong Zhong and Hua Hua are clones of the same macaque fetal fibroblasts. Cells from adult donor cells were also used, but those babies only lived for a few hours after birth.

"We tried several different methods but only one worked," says Sun. "There was much failure before we found a way to successfully clone a monkey."

The first author Liu Zhen, a postdoctoral fellow, spent three years practicing and optimizing the SCNT procedure. Including quickly and precisely removing the nuclear materials from the egg cell and various methods of promoting the fusion of the nucleus from the donor cell and enucleated egg. With additional help of epigenetic modulators that help re-activate the suppressed genes in the differentiated nucleus, he was able to achieve much higher rates of normal embryo development and pregnancy in the surrogate female monkeys.

"The SCNT procedure is rather delicate, so the faster you do it the less damage to the egg you have, and Dr. Liu has a green thumb for doing this," says Muming Poo, a co-author on the study, who directs the Institute of Neuroscience of CAS Center for Excellence in Brain Science and Intelligence Technology and helps to supervise the project. "It takes a lot of practice, not everybody can do the enucleation and cell fusion process quickly and precisely, and it is likely that the optimization of transfer procedure greatly helped us to achieve this success."

The researchers plan to continue improving the technique and monitoring Zhong Zhong and Hua Hua for their physical and intellectual development. The babies are currently bottle fed and are growing normally compared to monkeys their age. The group is also expecting more macaque clones to be born over the coming months. The genetically identical monkeys will provide a useful animal model to study human diseases with a genetic basis, such as cancer and immune disorders.

The lab is following strict international guidelines for animal research set by the US National Institutes of Health and encourages the scientific community to discuss what should or should not be acceptable practices when it comes to cloning of non-human primates. "We are very aware that future research using non-human primates anywhere in the world depends on scientists following very strict ethical standards," Poo says. There are no plans to conduct a similar study in humans.

This work was supported by grants from Chinese Academy of Sciences, the CAS Key Technology Talent Program, the Shanghai Municipal Government Bureau of Science and Technology, the National Postdoctoral Program for Innovative Talents and the China Postdoctoral Science Foundation.

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