In late November and early December, researchers at the Chinese Academy of Sciences Institute of Neuroscience witnessed something incredible: The birth of two genetically identical long-tailed macaques named Zhong Zhong and Hua Hua—a nod to the term ‘Zonghua,’ meaning the Chinese people. These monkeys are the first primates to be cloned using a technique called somatic cell nuclear transfer (SCNT), the same method that was used to create the first animal clone, Dolly the sheep, over 20 years ago.
The researchers at the Chinese Academy of Sciences are optimistic that the ability to produce genetically identical monkeys will allow unprecedented insights into human diseases, but the birth of Zhong and Hua is also a big deal for the science of cloning more generally. Primates have been notoriously resistant to cloning efforts over the past two decades due to the unique complexity of their cellular machinery. The birth of these macaque clones is a proof of concept that could lead to the cloning of other primates, and perhaps, eventually, humans.
"The technical barrier of cloning primate species, including humans, is now broken," Qiang Sun, the lead researcher on the project at the Chinese Academy of Sciences said during a press conference on Tuesday night. "In principle, it can be applied to humans. However, the reason we broke this barrier is to produce animal models that are useful for medicine and human health. There was no intention to apply this method to humans."
Hua Hua, the second SCNT primate clone. Image: Qiang Sun, et al. / Chinese Academy of Sciences
Somatic cell nuclear transfer is a type of reproductive cloning that involves taking the nucleus from any adult cell in the animal to be cloned and injecting it into the cell of a fertilized egg whose nucleus has been removed. In 1996, Dolly the sheep became the first animal to successfully be cloned using this method.
In the past two decades, 23 other species have also been cloned using SCNT, including cows, horses, cats, and pigs. The success of these experiments prompted fears that humanity was on the cusp of designer babies or the production of human clones for harvesting organs, which prompted President Clinton to issue a ban on the use of federal funds for human cloning research in 1997. As it turned out, however, making the jump from cloning sheep to cloning primates proved far more difficult than anticipated.
The first primate clone was technically a rhesus monkey named Tetra born in 1999. Tetra was produced by a process called embryo splitting, which is similar to how twins are born naturally. It involves dividing a two-cell embryo into two separate, identical embryos and allowing them to develop on their own. While this process is well documented and relatively straight forward, it limits the number of genetically identical offspring that can be produced from an embryo to a maximum of four. To get larger populations of clones, SCNT is necessary since it can use the nuclei from any number of adult cells of the animal to be cloned.
One of the main reasons why primates have been so resistant to SCNT cloning has to do with the way primate egg cells are structured. In unfertilized primate egg cells, proteins called mitotic spindles are clustered close to the cell’s chromosomes, unlike most other mammalian embryos, where the spindles are spread around the cell. These spindles are responsible for guiding chromosomes to the right place during the cell division process that eventually results in a live primate.
Over the past decade, however, researchers have found that when the nucleus—and hence all the chromosomes—of a primate egg cell is removed, this can also damage or rearrange the mitotic spindles. This means that when a new nucleus is injected into the embryo, the spindles are unable to guide chromosomes to the correct place during cell division. So while researchers were able to successfully create primate embryos on a number of occasions, errors during cellular division meant none of these embryos would last more than a few weeks in a primate’s womb.
Zhong Zhong and Hua Hua. Image: Qiang Sun, et al. / Chinese Academy of Sciences
"Numerous attempts have been made to clone nonhuman primate species," Sun said during a press conference on Tuesday evening. "They have all failed. This led to the idea that maybe the somatic nucleus of the primate species are unable to express the genes required for embryo development."
As detailed today in Cell, Sun and his colleagues at the Chinese Academy of Sciences surmounted this difficulty by optimizing the nuclear transfer process to minimize damage to the primate egg cell and adding some human RNA into the clone egg cell. This RNA essentially worked to turn on and off the genes that would otherwise inhibit the development of the primate embryo. It is, in Sun's words, "programming" the nucleus to express the genes that are required for embryo development.
Still, it took a while to get the programming right. According to the researchers, they tried using the nucleus from adult cells for clones in 42 monkeys, 22 of which became pregnant. Of these pregnancies, only two yielded live births, but both of the babies died a few hours after birth.
The researchers had better luck when they used the nucleus from connective tissue cells derived from an aborted monkey fetus, however. These clone cells were injected into 21 female macaques and resulted in four pregnancies. Of these, two pregnancies were aborted within two months, but the other two pregnancies resulted in the birth of two healthy, genetically identical long-tailed macaques. According to the researchers, the babies are still in excellent health almost two months later.
“We tried several different methods, but only one worked," Sun said in a statement. "There was much failure before we found a way to successfully clone a monkey."
According to Sun, the same method used to clone the macaques could be applied to humans, but he said it's highly unlikely that the government would allow its extension to human cells (besides the ethical controversy, the method also had a low success rates among the primate test subjects). Still, the SCNT method is a promising candidate for producing large populations of genetically identical primates, which Sun and his colleagues anticipate will be used to study human diseases while "greatly reducing" the number of animal subjects required to perform the studies.