Deft hands enable cloning
15 December 2006 (Volume 1 Issue 12)
In 1998, Teruhiko Wakayama, Team Leader of RIKEN's Laboratory for Genomic Reprogramming, surprised the world by achieving what had been considered impossible—cloning a mouse from somatic cells, or cells not involved naturally in reproduction. To date, only about 10 species of somatic cell clones have been produced. The success rate of cloning is low for all animal species, and animals that are born have abnormalities. This is considered attributable to imperfect programming of the nuclei of the cells transferred to egg cells. Since cloning technology is expected to be applied to regeneration medicine, elucidating the mechanism of genomic reprogramming is considered a big obstacle to overcome. The Laboratory for Genomic Reprogramming is now making full use of nuclear cell transfer technology with a micromanipulator, and is testing various conditions that potentially affect genomic reprogramming.
Birth of the world's first somatic cell cloned mouse
Wakayama headed the program that successfully led to the first birth of a mouse cloned from a somatic cell. It was in 1998 when he was studying abroad in Ryuzo Yanagimachi's Laboratory at the University of Hawaii. In the previous year, Dolly, the world's first sheep cloned from somatic mammalian cells was born.
“The birth of Dolly was shocking to me because everybody including myself took it for granted that it was impossible to create cloned mammals from somatic cells,” reflects Wakayama. The technology that enabled the birth of Dolly had been used for cloning fertilized eggs. Thus Dolly's birth was not a technical breakthrough, but a breakthrough in achieving what was previously thought impossible. Wakayama therefore thought he might be able to clone mice from somatic cells, which had also been considered impossible.
A clone is an organism that has the same genetic information as another organism or organisms. Clones are either 'fertilized egg clones' or 'somatic cell clones' depending on how they were produced. To create a fertilized egg clone a fertilized egg is taken from a female fallopian tube. After the fertilized egg has divided into eight cells, these cells are separated into eight individual cells. Then, the nuclei of the fertilized eggs are transferred to eggs that have had their nuclei removed. Finally the cloned eggs are returned to the womb. The first successful fertilized egg clone was a frog in 1952. Fertilized egg cloning technology has been applied practically in the animal industry since 1986 when scientists succeeded in cloning Dolly the sheep.
In contrast, somatic cell clones are produced by transferring nuclei obtained from various somatic cell tissues such as skin, milk gland, and cumulus oophorus to eggs that have had their nuclei removed in advance (Fig. 1).
Figure 1: How to create somatic cell cloned mice and ES cells of somatic cell origin.enlarge image
“No technical breakthroughs can be found in our somatic cell cloning. I guess we were just lucky,” says Wakayama. “I have been using a technique that uses a micromanipulator to remove the nucleus of an egg cell since when I was in Japan. At the University of Hawaii, I was engaged in micro-insemination with a micromanipulator, which is a technique to fertilize an egg by injecting a sperm into an egg under a microscope.” In the nuclear transfer procedure of somatic cell cloning, the nucleus of an egg is just replaced with the sperm used for micro-insemination. “Honestly, I just tried to combine a technique I already had and the piezo-drive technique, which happened to lead to successful somatic cell cloning,” he explains.
A micromanipulator is a device controlled by a joystick that enables accurate control of movement, often to an accuracy of less than 1 µm (Fig. 2). A piezo-drive is a driving mechanism with a pipette mounted on the tip of a piezoelectric element, which can be vibrated at a high speed by electricity. When a pipette is stuck into a cell, the cell usually collapses. However, using the piezo-drive, you can instantly make a hole through the cell membrane, and efficiently remove or inject a nucleus without collapsing the cell (Fig. 3). Of course, operating the micromanipulator, or the piezo-drive in particular, is not easy, and requires well-practiced skills.
Figure 1: Micromanipulator system. Enucleation and nuclear cell transfer (Fig. 3) are conducted by operating the joystick of the micromanipulator and the injector by hand and the switch of the piezo-drive by foot, and confirming the images observed through the microscope and listening to the changes in the operating sounds produced by the piezo-drive. Operating this system requires extreme concentration and well-practiced operational skills.enlarge image
Successful somatic cell cloning with mice has great significance. It was viewed as a supplementary experiment to sheep cloning, because doubt had been cast on whether or not Dolly was a somatic cell clone. Further, since mice are common experimental animals, it allowed cloning research to start in earnest.
Success in improving the success rate of mouse cloning
In 2002, Wakayama established the Laboratory for Genomic Reprogramming at RIKEN Center for Developmental Biology. “Our biggest challenge is to improve the success rate of mouse cloning from somatic cells,” says Wakayama.
The success rate of somatic cell cloning is very low. For example, in the first experiment with sheep cloning, 227 nuclei were transferred to egg cells, but only Dolly was born. Thus, the success rate was about 0.4%. In Wakayama's experiments with mice, the rate improved to around 2%. Cows and goats cloned from somatic cells were also produced using this technique, but the success rate was in the range of 2–10%. Unfortunately, each individual had some kind of abnormality, such as obesity or a breathing disorder, and some died young. The key to understanding the cause of these problems is a process known as 'reprogramming'.
Reprogramming is the process of regenerating the nucleus of a cell that is differentiating into various cells. That is, scientists return the nucleus to its anaplastic state at fertilization. “Eggs and sperm are specialized cells, in which differentiation has developed. However, when an egg is fertilized, the nucleus is reprogrammed so that the fertilized egg can form into a new individual,” explains Wakayama. Imperfect regeneration of the nucleus transferred to an egg is considered to be the cause of the low success rate of somatic cell cloning and the presence of abnormalities. “If the nucleus were perfectly reprogrammed, the abnormalities would disappear,” he notes. “However, we do not understand at all how the nuclei of egg or sperm cells are reprogrammed.”
Scientists all over the world are conducting research into the reprogramming mechanism of nuclei. Although many scientists are adopting molecular biological approaches used for investigating the expression of genes and functions of proteins, Wakayama is taking a completely different approach. His team has the world-leading micromanipulating technique so his plan is to take advantage of this technique, and produce many somatic cell clones. Conducting experiments under various conditions and investigating the changes in the success rate may lead to the discovery of key factors related to genomic reprogramming he says.
In 2005, the Laboratory for Genomic Reprogramming succeeded in improving the success rate of cloning mice from 2% to 6%. Wakayama explains that the condition the team varied was the addition of a chemical called trichostatin A to the culture solution of cloned embryos—and it served to enhance the genomic reprogramming. “This was the first example that clearly improved the success rate since the research into somatic cell cloning started, and it was expected to add a fresh dimension to our understanding of the mechanism of genomic reprogramming.”
The Laboratory for Genomic Reprogramming was successful in achieving this breakthrough due to Satoshi Kishigami, a research scientist at Wakayama's laboratory, who determined the optimum conditions for genomic programming in which condensation and processing time are the two most important factors. “In the search for factors related to genomic reprogramming, we need to tenaciously try to test various conditions,” says Wakayama. Thus, a large number of nuclear-transferred egg cells are needed. His team is capable of transferring about 1000 nuclei a day. “This number is amazing because no such capabilities in laboratories have so far been reported in the world,” says Wakayama firmly. There are only four laboratories in the world (including Wakayama's) in which somatic cell cloned mice can be produced.
Figure 3: Transfer of a nucleus to an enucleated egg.enlarge image
Expectations for ES cells of somatic cell origin
The team has further improved the success rate of cloning mice from somatic cells. Wakayama looked to ES cells, or embryonic stem cells of somatic cell origin to reliably create cloned mice. Instead of returning cloned embryos to the uterus, the team cultured them until they developed into blastocysts, thus creating ES cells (Fig. 1). Although the success rate for somatic cell clones was 6%, the rate for ES cell-based clones increased to around 50%. “Once created, ES cells can increase in large numbers,” explains Wakayama. “Thus, in theory, we can create an infinite number of clones by using their nuclei.”
With a degree in agriculture, Wakayama is interested in applications to the animal industry. “How nice it would be, if the research into mouse cloning which I am conducting now could benefit society, and as a result I could have the chance to enjoy delicious Kobe beef at a reasonable price,” he muses.
Wakayama also acknowledges potential medical applications of his work since ES cells can differentiate into any type of cell. If ES cells are created from somatic cells taken from a patient, and returned to the patient's body after they have differentiated into necessary cells or tissues, the patient can receive medical treatment without any immunologic rejection reaction. Fertility treatment will also be available if egg or sperm cells are created from ES cells. “In our laboratory, we created ES cells from the tail cells of a sterile mouse that could not create sperm cells, and made the ES cells differentiate into sperm, which were then used to fertilize egg cells, thus successfully creating a cloned mouse.”
Challenging the limits
Whenever possible, Wakayama welcomes people who want to learn nuclear transfer technology. “I myself learned various techniques from others,” he says. “Thus it is very natural for me to teach this newly developed technique to others. Furthermore, if someone other than me fails to reproduce what I have done, that person will consider it to be a lie. I think an engineer finds it nice to see the technology he developed popularized.”
To explain why he has an illustration of the solar system next to his own micromanipulator, Wakayama says: “I wanted to be an astronaut. I once applied for the astronaut program; I flunked the English test, though. From my childhood, I was a big fan of science fiction and I always dreamt of going into space. I remember I was very excited to see the word 'clone' in a science fiction story. My dream of going into space did not come true, but I have the feeling that I have been doing what I love to do and so, as it turned out, I find myself here.”
And, his dream for the future? “Developing new technology that can make a significant difference even in what seemed impossible. I would like to challenge the limits in the field of technology.”