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For Immediate Release:
July 22, 1998
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First Reproducible Cloning of Mammals from Adult Cells Reported in July 23 Issue of the journal Nature
The first reproducible cloning of a mammal from adult cells, which has successfully yielded three generations and more than 50 identical cloned mice, is reported in the July 23 issue of the international science journal Nature by an international team of scientists, lead by Ryuzo Yanagimachi, of the University of Hawaii.
The distinctive cloning technology, described as the Honolulu technique, could be more viable for the production of drugs using transgenic animals than earlier techniques because of its efficiency of reproducibility and, when used in genetic and embryonic development studies, will shed new light on the cellular and molecular activities involved in aging and diseases such as cancer, AIDS, diabetes and multiple sclerosis. The technology has been licensed to the Hawaii-based biotechnology company ProBio America, Inc., for commercialization and to test it for expanded uses.
The investigators anticipate that due to similarities between development in mammals the technique will be applicable to larger animals. For example, efficient and accurate cloning can improve the reliability and safety of reproducing transgenic mammals, such as cattle, pigs and sheep, that can be used in the economical production of lower cost protein-based pharmaceuticals. The technique may also be useful for cloning wild or endangered species in a controlled environment.
"Our study validates animal cloning, which we did using an injection method and adult cells. Our method differs substantially from previous techniques. Earlier procedures generated clones either by injection or fusion of embryonic or fetal cells or by the fusion of adult cells, which is how the sheep Dolly was created," explains Dr. Yanagimachi, the paper's senior author. He is a professor in the Department of Anatomy and Reproductive Biology at the John A. Burns School of Medicine of the University of Hawaii at Manoa, located in Honolulu.
In the experiment reported in Nature, the scientists used adult mouse cells to create new mice that are genetically identical to the parent mouse. Teruhiko Wakayama, a postdoctoral researcher working in Dr. Yanagimach's laboratory, pioneered the Honolulu cloning technique.
Using a special pipette, the donor nucleus is microinjected into an egg whose nucleus was previously removed. The researchers cultured the resulting cell, placed it in a surrogate mouse and allowed the clone to develop. By repeating the procedure, the team created second and third generations of cloned mice that genetically match their sister/parent, sister/grandparents and sister/great grandparent.
"We succeeded both in using a new method and new cell type to clone mice from adult cells and in repeating it to produce clones of clones of clones-essentially identical mice born a generation or more apart," said Dr. Wakayama, the study's lead author.
The donor nuclei came from cumulus cells, which surround developing eggs within the ovaries of female mice. Each nucleus contains all of the genetic instructions needed to create an adult, even though specialized adult cells do not need, or use, all of the instructions to exist. In contrast, embryonic cells have not yet specialized into their adult fates and, therefore, are still using many of their genetic instructions.
"We had to turn back the clock of an adult cell so that it behaves like a newly fertilized embryo, which would develop into a normal adult," says co-author Anthony Perry, a postdoctoral researcher in Dr. Yanagimachi's laboratory.
Within five minutes of the donor nucleus removal, the researchers inserted it into the developing egg cell, called an oocyte, using the special injection pipette. The oocytes removed from adult female mice had already undergone the first part of their two-step maturation process. The second step typically occurs with the stimulation of a fertilizing sperm.
In the study, the insertion of the donor nucleus preceded the second maturation step, and the scientists delayed this maturation anywhere from one to six hours. This delay increased the likelihood that when the oocyte continued its maturation, a process called activation, it would divide and develop normally.
After activation, the cells divided repeatedly to reach the multi-cell stage at which an embryo is called a blastocyst. Cells in blastocysts begin to mass in preparation to form the first tissues of an embryonic mouse.
"We discovered that a relatively high proportion of the oocytes developed into blastocysts and then further developed when we included a delay between the nuclear injection and the oocyte activation," explains Dr. Yanagimachi. "The exposure after injection of the donor nuclei to the oocyte cytoplasm, which is so rich in the factors that promote cell division, appears to facilitate the nuclear changes essential for development. We will study the molecular events of this delay period in future work."
Quartet of Experiments Verifies Technique
In a series of four experiments, the team examined the development of the transplanted blastocysts in surrogate female mice.
First, they transferred 142 blastocysts into 16 surrogates. Between 8.5 and 11.5 days after nuclear insertion (dpc), the scientists found five live and five dead fetuses.
In the second experiment, the research team placed 800 blastocysts into 54 foster mothers. Cesarean sections at 18.5 and 19.5 dpc revealed 17 live fetuses. Of these, 10 survived, six died after delivery and one died a week later. All of the surviving offspring, including the first born named Cumulina, grew into adults, were mated and delivered and raised normal offspring. Several of the offspring have now been tested and are confirmed to be fertile adults.
In the third experiment, the scientists proved genetically that the offspring produced in the studies were clones. By injecting nuclei from agouti mice, whose coats are coffee-colored, into the oocytes of genetically black mice, the researchers produced mice with the agouti coloring. The scientists verified the clones were pure agouti mice by performing a genetic analysis of their placental tissue. Of the 298 blastocysts transferred to 18 albino (white) foster mothers, six developed by 19.5 dpc into live fetuses. All but one lived.
"Our DNA typing results provided substantiating evidence that the offspring were indeed clones and that their genetic composition was identical to the cumulus cell donor females and did not contain DNA derived from either oocyte donors or host foster mothers," says Kenneth R. Johnson, research scientist at the Jackson Laboratory in Bar Harbor, Maine, who also contributed to the research.
Clones produced in this third experimental series were themselves used as nucleus donors in a fourth set of experiments. These experiments showed, perhaps for the first time in any species, that clones can be made from clones.
Overall, Yanagimachi and his team found that high blastocyst implantation rates of up to 71 percent yielded development rates of fetuses at 516 percent and of full term mice at 23 percent. The authors note this work clearly confirms that mammals can be reproducibly cloned from adult cells.
This nuclear transfer technique provides a ready model for researchers. Specifically, scientists can use cloned mice to evaluate the molecular mechanisms that regulate the reprogramming of adult cell genetic material and the influence of genes and their activation during embryonic development.
"Access to cloned laboratory mice, whose genetic development is known, permits, for example, such studies as the role of a given gene in the developing body or in the process of disease," says Dr. Perry. "We really know very little about the mechanism of early development, and this cloning technique should help us to learn much more."
The National Institute of Child Health and Human Development (NICHD), part of the U.S. government's National Institutes of Health, and ProBio America, Inc., funded the research in part. The Japanese Society for the Promotion of Science and the European Molecular Biology Organization provided fellowship support.
"This is a very important advance which has illuminated some of the fine points of the technology that need to be mastered in order to make nuclear transfer technology efficient," says Michael E. McClure, chief of the Reproductive Sciences Branch of NICHD. "This is a case where the right cell type, the right research design, and the right technical expertise came together with great success."
Yanagimachi's co-authors from the University of Hawaii include Drs. Wakayama, Perry and Maurizio Zuccotti. Wakayama also has an appointment at the Department of Veterinary Anatomy at the University of Tokyo in Japan. Zuccotti, a postdoctoral researcher in Yanagimachi's laboratory, has an appointment at the Dipartimento Biologia Animale at the University of Pavia in Italy. Perry also has a senior fellowship in the Department of Signalling at the Babraham Institute in Cambridge, England.
The University of Hawaii is the state's 10-campus system of public higher education. The 17,000-student main Manoa campus is a Carnegie I research university of international standing that offers an extensive array of undergraduate, graduate and professional degrees. The University's research program, which draws $160 million in extramural funding annually, is widely recognized for its strengths in tropical medicine, evolutionary biology, astronomy, oceanography, volcanology, geology and geophysics, tropical agriculture, electrical engineering and Asian and Pacific studies. Visit UH at www.hawaii.edu.
The Jackson Laboratory, founded in 1929, is a world leader in mammalian genetics research. With more than 850 employees, the nonprofit, independent facility has a threefold mission-to conduct basic genetic and biomedical research, train present and future scientists and provide genetic resources to researchers worldwide. Information is accessible at www.jax.org.
ProBio America, Inc., is a Honolulu-based biotechnology company whose aim is to assist in the development and commercialization of reproductive and DNA preservation research for pharmaceutical and agricultural purposes. ProBio has acquired licensing rights and research funding obligations from the University of Hawaii and holds patents in the area of mammalian research and for technologies in the related fields. See information at www.probioinc.com.
Scientists Report First Reproducible Mammalian Cloning from Adult Cells of Adult Mice in the International Science Journal Nature
- A Contextual Backgrounder -
A clone is most simply defined as an exact copy-a genetic replica. Cloning refers to the generation of multiple copies of a gene, a cell or an entire organism. Whereas gene cloning and the manipulation and transfer of genetic material within and across species has been a scientific reality for more than two decades, cloning complete organisms, and in particular mammals, has long been the a topic of science fiction thrillers, shrouded in futuristic mystique. With the recent arrivals of Dolly, Polly and Gene-two lambs and a cloned calf-the future is here, and researchers are just now beginning to explore beginning to exploring the potential scientific and commercial applications of the technology.
With each cloning success scientists seek to duplicate their findings and refine their techniques, further defining the molecular mechanisms of replication, gene activation and development involved in the cloning process. In the making of Dolly, researchers at the Roslin Institute, outside Edinburgh, Scotland, produced 277 genetically reprogrammed eggs, but only one survived and developed into a viable lamb. For cloning to realize its commercial potential, the technique had to become more efficient-veritably a standard laboratory procedure.
The research published in the July 23, 1998, issue of Nature by a scientific team lead by Ryuzo Yanagimachi, of the John A. Burns School of Medicine at the University of Hawaii in Honolulu, describes the first successful mammalian cloning from adult mouse somatic cells using a technique called nuclear transfer by microinjection. In contrast, Dolly, like other recently described clones, was created via by a different method, elctrofusion, using mammary-derived cells from adult sheep. Dr. Yanagimachi's collaborators in the effort to clone adult mice included co-authors from the University of Hawaii at Manoa (in Honolulu), the University of Tokyo (Japan), the University of Pavia (Italy), the Jackson Laboratory (Bar Harbor, Maine) and the Babraham Institute (Cambridge, UK). ,
The team's technique, which has been licensed to the biotechnology company ProBio America, Inc., provides scientists a valuable tool to for the create creation of model systems useful for evaluating and controllingthe molecular mechanisms that influence the genome and for the study of to study embryo formation and cell differentiation. The method, known as the Honolulu technique, may be more useful than earlier techniques because it affords the researchers greater ability to manipulate the adult donor nucleus. This ability will have application industry-wide in increasing the study of the role genes play in aging and the disease processes.
Dolly, the first mammal cloned from an adult cell, stunned the scientific community when she was introduced in the February 27, 1997, Nature as a 7-month-old lamb. Animal cloning was, however, not a revelation. For decades, animal breeders have divided young embryos and coaxed clumps of these embryonic cells to give rise to fully formed animals, a process called embryo splitting. Each of the two, three or more offspring that at result from the splitting of a single embryo share the same genetic makeup, as do naturally formed identical twins. Embryo splitting is possible because young embryonic cells share an attribute called totipotency. The cells have not yet begun to differentiate into a specialized cell types (a kidney, skin or eye cell, for example); they still have the potential to become any and every type of cell in the adult animal.
Furthermore, scientists have long performed the nuclear transfer and electrofusion techniques used in the creation of Dolly. The process involves removing the nucleus of an unfertilized egg, in this case a sheep's egg, and retaining only the egg's cytoplasm. A nucleus isolated from a mammary derived cell of the sheep animal to be cloned was then inserted into the egg. This nucleus contains the DNA that will determines the sex, genetic traits and physical characteristics of the cloned animal. The clone is virtually genetically identical to the adult sheep that donated the nuclear material, with the one exception being the of small amounts of mitochondrial DNA that remain in the cytoplasm of the egg.
Before Dolly, nuclear transfer was only successful when the nucleus came from an embryonic or fetal cell. Attempts to clone animals, including mice, from adult cells failed. Scientists assumed that adult cells, because they had already differentiated and lost their totipotency, would require some sort of molecular rewiring before they could regain the developmental capabilities of embryonic cells.
The Roslin group appears to have achieved this genetic reprogramming by starving the adult sheep cells before harvesting their nuclei. Depriving the cells of nutrients induces their genes to enter an inactive stage, ceasing growth and cell division. Once the adult nucleus is inserted into the egg, reactivation of the genetic material led to healthy, dividing embryos. The embryo that became Dolly was implanted and carried to term in a surrogate mother sheep. Alternatively, these embryos can be used to repeat the cloning process and produce a second generation of clones. Dolly has since demonstrated her reproductive capability, producing sexually derived offspring of her own.
The cloning of adult mice, reported by Drs. Wakayama, Yanagimachi and their colleagues, was accomplished using nuclei from adult cumulus cells, which surround the developing ovarian follicle in female mice. The scientists selected these cells partly because they typically exist in a nondividing, quiescent state, making them more amenable to genetic reprogramming. The cumulus nuclei were injected with a special pipette into enucleated mouse eggs, yielding 800 embryos in one series of experiments using a special pipette. Of these, 17 developed fully, and of the implanted embryos, 10 healthy female mice survived, including the first, Cumulina, and all and each have produced their own offspring. In another experiment, the eggs of black mice were the recipients of nuclei from coffee-colored agouti mice. Of the 298 embryos implanted in albino surrogates, five mice survived, and all shared the agouti coloring of their genetic parent. Overall, the scientists created more than 50 clones.
Following closely on Dolly's success came Gene, a cloned calf. Perhaps of greater commercial interest, though, was Polly, the first cloned transgenic lamb. Polly was cloned from fetal sheep fibroblast cells that had been genetically engineered to contain the human gene for factor IX protein, a blood clotting agent used to treat hemophilia. Although not derived from an adult cell, Polly is proof of principle that a viable animal can arise following genetic manipulation of the donor's DNA.
The advent of recombinant DNA technology in the early 1970s gave scientists the means to insert a foreign gene into the genome of a host organism-to clone a gene. In 1982, the commercial potential of this technology was first realized when the biotechnology company Genentech brought to market recombinant human insulin. Recombinant human growth hormone, beta-interferon and the blockbuster drug erythropoietin (epo) soon followed. These drugs are all produced by the transfer of human genes into bacterial or mammalian cells, which are then grown in large-scale culture systems to produce commercial quantities of the therapeutic proteins.
Foreign genes can be inserted into mammalian embryos, typically by microinjection, yet it is an inefficient, hit-or-miss technique. Creating a transgenic "founder" animal that carries the acquired trait in its germ line and can pass it on to its progeny requires a great deal of time and expense. The ability to clone such founder animals to create transgenic herds for large-scale drug production offers the industry a more efficient alternative.
Some human proteins that might have value as pharmaceuticals or nutritional supplements are not amenable to economical production in bioreactors, as they require complex modifications that cannot be carried out by bacterial cells. One attractive alternative, dubbed "pharming," is to the transfer of these genes into livestock. By expressing the target genes selectively in the mammary glands of lactating goats, sheep or cows, for example, researchers can then purify the desired protein products from the large quantities of milk produced by herds of these transgenic animals.
In the laboratory, transgenic animals, such as mice and rats, are invaluable to medical researchers. Scientists can study the function of uncharacterized genes and can evaluate their role in biochemical pathways and in disease processes. By inserting a known pathogenic gene into a laboratory animal, researchers can create animal models of human diseases. These models are powerful and preferred tools for understanding disease, for identifying novel therapeutic targets and for evaluating the effectiveness of experimental therapies. Based on the work in mice and some of the novel features of the Honolulu cloning technique, it should be possible to rapidly advance the method for use in large animals.