Scientists In Hawaii Report First Reproducible Cloning Of Mammals From Adult Cells

Honolulu Technique Could Be More Viable Means For The Production Of Drugs -- Clones Can Be Made Of Clones

HONOLULU, July 22, 1998 -- 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 Nature by an international team of scientists, lead by Ryuzo Yanagimachi, Ph.D., 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 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.

In the experiment reported in the July 23 Nature, the scientists used adult mouse cells to create new mice that are genetically identical to the parent mouse. Teruhiko Wakayama, Ph.D., a postdoctoral researcher working in Dr. Yanagimachi'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. However, 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, Ph.D., 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, Ph.D., 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, Dr. Yanagimachi and his team found that high blastocyst implantation rates of up to 71 percent yielded development rates of fetuses at 5 to 16 percent and of full term mice at 2 to 3 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, Ph.D., 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."

Dr. Yanagimachi's co-authors from the University of Hawaii include Drs. Wakayama, Perry and Maurizio Zuccotti, Ph.D. Dr. Wakayama also has an appointment at the Department of Veterinary Anatomy at the University of Tokyo in Japan. Dr. Zuccotti, a postdoctoral researcher in Dr. Yanagimachi's laboratory, has an appointment at the Dipartimento Biologia Animale at the University of Pavia in Italy. Dr. 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 more than $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.

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 about The Jackson Laboratory is accessible via the World Wide Web 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. More information on the company is available at http://www.probioinc.com.

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Editor's Note: The original news release, with links to accompanying graphics and other supporting materials, is available at http://www2.hawaii.edu/~ur/News_Releases/NR_July98/cloning.html or (for journalists) through http://www.noonanrusso.com/news/hawaii/98news/Media/entry.ihtml