(Translated by https://www.hiragana.jp/)
PNAS Classics -- Nuclear Transfer
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Nuclear Transfer: Bringing in the Clones



by cbrownlee{at}nas.edu
PNAS Staff writer


Removing an egg's nucleus during nuclear transfer. Courtesy of Roslin Institute, Edinburgh.
Put aside those sci-fi comic books and pick up a newspaper—the future of cloning is now. From the Raelian's heavily hyped (and likely erroneous) claims of human cloning to the recent (and very real) advances in so-called therapeutic cloning, scientists, journalists, and ethicists have riled the public with each novel advance. Despite the recent buzz over this tantalizing technology, the basis for most cloning procedures—a technique called nuclear transfer—has been around for over fifty years.

Nuclear transfer is a two-part process: first, scientists remove the nucleus from an egg, and second, they replace it with the nucleus of an older donor cell. A new clone—a genetic copy of the donor—forms when the egg starts to divide. Despite the seemingly simple nature of this technique, successful nuclear transfer thwarted scientists for many years after it was first proposed in 1938 (1). However, thanks to the patient efforts of two scientists, Robert Briggs and Thomas King, nuclear transfer was finally accomplished with the frog species Rana pipiens. Their findings were published in a 1952 PNAS paper (2), irreversibly expanding the fields of genetics and development and setting the stage for current cloning efforts.


 
A Fundamental Question


John Gurdon (1933-). Courtesy of J.G.
The original motivation for nuclear transfer experiments related to a question that had bantered around the scientific community for decades: what happens to the genome as a fertilized egg, or zygote, develops into an embryo? A zygote is totipotent, capable of forming every type of body cell. As the zygote divides, the cells differentiate and become specialized to perform a specific set of functions, such as skin, bone, or intestinal cells. Scientists had two opposing theories on how cells became differentiated: the cells either lost all genes except those relevant to their specific task, or they kept the entire genome and selectively turned genes on or off.

"To me, it was an important fundamental question in development—the problem was how to answer that question," said Sir John Gurdon, Academy member and professor of cell biology at the University of Cambridge.

The experiment that would provide an answer was first envisioned by Hans Spemann, an embryologist and Nobel laureate who won the 1935 Prize in Medicine for developing new embryological surgery techniques. Spemann reasoned that if an egg implanted with a nucleus from a differentiated cell still developed into a normal embryo, this would prove that the nucleus retains a full genome capable of directing all types of differentiation. In other words, a differentiated nucleus could still be totipotent.

Hans Spemann (1869-1941). Courtesy of the Max Planck Society Friedrich Miescher Laboratory.
In 1938, Spemann published an account of his experiments with a rudimentary nuclear transfer technique (1). Using a piece of hair to constrict a newly fertilized salamander egg, Spemann separated the egg's nucleus on one side and the cytoplasm, or other cellular material, on the other. After the nucleated side divided 4 times, creating a 16-cell embryo, Spemann loosened the hair and allowed a nucleus to slip back into the separated cytoplasm. Cell division now began on this side as well, and by tightening the hair loop again, Spemann broke apart the two embryos. The end result was a twin set of salamanders, one slightly younger than the other.

Although this work showed that the nucleus remained totipotent after undergoing four divisions, Spemann wondered whether nuclei from much older embryos, or even adult organisms, had similar potential. Transplanting an older nucleus into an egg would be a "fantastical experiment," wrote Spemann. However, for the next 14 years, scientists struggled with making such an experiment work.
Fantastical Experiment or Hare-Brained Scheme?


Robert Briggs (1911-1983).Reprinted with permission from Biographical Memoirs V. 76 ©(1999) by the National Academy of Sciences.
The idea of nuclear transfer eventually caught the attention of Robert Briggs, an imaginative character who'd worked in a shoe factory and earned money as a banjo player before becoming a scientist (3). In 1949, then a researcher at Lankenau Hospital Research Institute in Philadelphia (now known as Fox Chase Cancer Center), Briggs was studying the role of the nucleus and its chromosomes in development. His previous studies centered mostly on chromosomal abnormalities, such as haploidy and triploidy, but Briggs was completely unaware of Spemann's earlier experiments.

Briggs' funds would not support his new interest in nuclear transfer research, so he applied for a grant with the National Cancer Institute (NCI) in 1949. "Really, all he wanted was a stipend for a research fellow and some money for supplies and equipment. It was a modest grant, even in reference to those days," said Marie DiBerardino, Academy member, former research assistant with Briggs and King, and professor emerita at Drexel University.


However, Briggs' first grant application was immediately turned down. In the ensuing correspondence, NCI called nuclear transplantation a "hare-brained scheme" with little chance of success (4). Although disappointed, Briggs did not give up. He reapplied, insisting on a site visit by an NCI representative. Taking the representative on a tour of the lab may have been the ticket Briggs needed—his second application was approved, giving him enough money to hire an assistant.
A Clever Method


Thomas King (1921-2000). Courtesy of the Society for Developmental Biology.
To find the right research partner, Briggs relied on the expertise of Roberts Rugh, a colleague and well-known embryologist at Columbia University. Rugh knew the perfect candidate for the job—Thomas King, who had trained extensively in microsurgical techniques. Such proficiency would be essential in performing the minute manipulations necessary for nuclear transfer. At the time, King was attending graduate school at New York University after serving in the United States Army.

"[King] was married, he had a little girl, and he was working like mad teaching at night just to support himself and his family. He saw this as a good opportunity," said DiBerardino.

King relocated to Philadelphia, and the two scientists immediately got to work establishing a procedure for nuclear transfer. Although the basic protocol for removing an egg nucleus had been developed over a decade prior (5), the logistics of re-implanting a nucleus donated by another cell were not yet known. "The real challenge here was to not kill the nucleus when you're injecting it," said DiBerardino.

Stages of nuclear transfer. The nucleus is removed from an egg (yellow photos) and replaced by a nucleus from a donor cell (blue photos). Courtesy of Roslin Institute, Edinburgh.
Briggs and King increased their chances of success by working with Rana pipiens, a North American species of leopard frog. Like many other amphibian species, Rana pipiens' eggs are easily harvested and, at more than a millimeter wide, relatively simple to manipulate. "They're huge, compared to other eggs," said Gurdon. "Chickens don't count because most of the egg doesn't form an embryo. So, in terms of complete eggs that form embryos, amphibian eggs are really quite unique."

To obtain a donor nucleus, King isolated a single cell from an embryo in the blastocyst stage. At this point, the embryo consists of a few thousand cells clumped together in a hollow ball. Using a tiny glass needle, or pipette, that was only slightly smaller than the cell, King applied gentle suction. With just the right touch, the cell broke around the outside of the pipette, allowing the nucleus to be drawn inside unharmed. This donor nucleus was then inserted into an egg from which the nucleus had been removed.

"The method of sucking a cell into a pipette was a clever one because they got in just one nucleus," said Gurdon. "If you put in 100 nuclei, it doesn't work."
Confusion in the Field


Northern leopard frog (Rana pipiens). Photo by Jeff LeClere, courtesy of Iowa Herpetology.
Despite its tedious nature, Briggs and King perfected their technique, and spent two years amassing a bevy of results from nuclear transplantation experiments in Rana pipiens. Reliably, around 40% of the eggs with donor nuclei resulted in the development of embryos, and later, tadpoles. The experiments were deemed a rousing success—finally, there was proof that older nuclei could direct the differentiation needed to create an embryo. The two scientists published their work in PNAS in March of 1952 (2), garnering the respect and attention of others in the field.

Briggs and King quickly moved on to the next question: does a cell's nucleus remain totipotent as it continues to age and differentiate? After all, cells in the blastocyst stage are still relatively early in development. It was unclear whether nuclei from older, more specialized cells would retain the ability to direct embryonic development.

Briggs and King attempted to perform nuclear transfers with progressively older cells, and found that as the cells developed it became much more difficult to produce clones (6). These results led the scientists to conclude that genetic potential diminishes as cells differentiate, and that it is impossible to produce a clone from the nucleus of an adult cell. However, John Gurdon and his colleagues at Oxford University produced conflicting research, published in a 1958 paper (7). Using Xenopus laevis, another species of frog, Gurdon's team showed that nuclei from fully differentiated intestinal cells could produce clones.

"There was a lot of confusion in the field for awhile," said DiBerardino.

Dolly the sheep (1996-2003). Courtesy of Roslin Institute, Edinburgh.
The question remained unanswered decades later, even after high profile cloning successes including Dolly the sheep, cc the "copy cat" (8), and a guar (an endangered ox-like animal) named Noah, among others. Although cells from adult donors produced these animals, scientists had no way of knowing whether the donated cells were fully differentiated or actually stem cells—undifferentiated cells capable of producing many cell types.

In 2002, scientists from the Whitehead Institute resolved the debate by cloning mice from the nuclei of adult lymphocytes, a type of immune system cell (9). Lymphocytes have genetic rearrangements that distinguish them from the body's other cells; thus, the donor nuclei in the study came equipped with a built-in marker system, automatically setting them apart from stem cells that may have contaminated the samples. The scientists were able to see direct evidence that the clones came from differentiated cells—each bore the distinctive mark carried by its donor's lymphocytes.

These results confirm that it is possible to produce a clone using the nucleus of a fully differentiated cell; however, it certainly isn't easy. "Forty percent is an extraordinarily high success rate," said Gurdon, referring to Briggs and King's results with nuclei from blastocyst cells. "If you take the nuclei of adult animals, they work at less than one percent." Difficulties specific to mammalian cells resulted in 276 unsuccessful tries before Scottish researchers produced Dolly (10). Some species, such as primates, dogs, and chickens, have thus far proven intractable to cloning (11).
Ethical Dilemmas


Limited success rates notwithstanding, cloning research continues to advance at a rapid pace. Briggs and King's 1952 speculation that nuclear transfer "may have other uses" seems unbelievably timid, given the panoply of cloning technologies envisioned by today's scientists. Reproductive cloning—creating genetic copies of an entire organism—could be used to reliably reproduce animals genetically engineered to produce drugs or with organs suitable for transplantation into humans. Reproductive cloning might also help endangered species avoid extinction, or perhaps even bring back extinct animals. Therapeutic cloning—creating embryos to harvest stem cells—could be used to produce human organs for transplant or to treat degenerative diseases like Alzheimer's or Parkinson's.

Inevitably, as cloning technology becomes more sophisticated, ethical questions loom large. Reproductive cloning is not a risk-free procedure. "In a number of species, animals have turned out not to be completely normal, even if it's subtle—like the rate at which they age, or how their immune systems function. It turns out that Dolly, in fact, was not [normal]," said Beatrice Mintz, Academy member, former graduate student of Robert Briggs, and researcher at Fox Chase Cancer Center. Research published in 1999 (12) suggested that the genetic material in Dolly's cells was aging prematurely. Although sheep can live to the age of 11 or 12, Dolly was only six and a half years old at the time of her death in 2003. Scientists have also found evidence of genetic abnormalities in cloned mice (13). Uncertainty regarding the safety of current cloning techniques led the authors of a 2002 National Academies report (14) to recommend a legal ban on human reproductive cloning.

As for therapeutic cloning, "the chief ethical objection is that the combination of a transplanted somatic [bodily] nucleus and an unfertilized egg constitute a potential human being and should not be used as a source of spare parts," noted Gurdon and co-author James Byrne in a recent PNAS perspective on nuclear transfer (15). "However," the authors pointed out, "in the absence of implantation, a reconstituted embryo has no possibility of becoming a human." Confusing the issue even further, scientists in China claim to have created cloned embryos by fusing a human donor cell nucleus with a rabbit egg cell, thereby blurring the boundaries between species (16). Advocates of cloning point to the potential medical benefits of such research, but each new advance carries a host of religious and ethical quandaries unlikely to be settled in the near future.

In all these controversies, it's easy to lose sight of the original purpose of cloning: to answer a fundamental question about the fate of genes during development. Briggs and King's 1957 paper began the quest for a solution and provided a set of tools for future research.

"Somehow an egg knows how to turn into a complete individual, with no instructions from anyone. How can it be that all the different cells arise in the right place from the same initial set of genes? Briggs and King were the first people to set about solving that amazing process," said Gurdon.
References


1. Spemann, H. (1938) Embryonic Development and Induction (Yale University Press, New Haven).

2. Briggs, R. & King, T. J. (1952) Proc. Natl. Acad. Sci. USA38, 455-463. |Article|

3. National Academy Press Biographical Memoir |Article|

4. Di Berardino, M. A. and McKinnell, R. G. (2001) Differentiation67, 59 - 62. |Article|

5. Porter, K.R. (1939) Biol Bull Woods Hole77, 233–257.

6. Briggs, R. & King, T. J. (1957) J. Embryol. Exp. Morphol.100, 269 - 312.

7. Fishberg, M., Gurdon, J. B., & Elsdale, T. R. (1958) Nature181, 424.

8. Westhusin, M. et al. (2002) Nature415, 859. |Article|

9. Hochedlinger, K. & Jaenisch, R. (2002) Nature415, 1035 - 1038. |Article|

10.Wilmut, I. et al. (1997) Nature385, 810-813.

11. Human Genome Project Fact Sheet

12. Shiels, P.G. et al. (1999) Nature 399, 316 - 317. |Article|

13. Jaenisch, R. et al. (2002) Proc. Natl. Acad. Sci. USA99, 12889 - 12894. |Article|

14. Committee on Science, Engineering, and Public Policy, Board on Life Sciences (2002) Scientific and Medical Aspects of Human Reproductive Cloning (National Academies Press, Washington, DC). |Report|

15. Dennis, C. (2002) Nature419, 334 - 336. |Article|

16. Gurdon, J.B., & Byrne, J.A. (2003) Proc. Natl. Acad. Sci. USA100, 8048-8052. |Article|
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