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Mice Join Clone Club

By Sean Henahan, Access Excellence

Honolulu, HI (7/22/98)- A lab full of Hawaiian mice have become the latest members of the small club of animals cloned from adults cells.

Researchers at the University of Hawaii announced the cloning of 22 healthy and fertile female mice which were cloned from nuclei of adult ovarian cumulus cells.  As in the famous and controversial case of Dolly the sheep clone, the scientists used donor cell nuclei that were in the 'quiescent' G0 stage of the cell division cycle. The cloned mice derived from nuclei from cumulus cells, ovarian cells that surround the egg and are shed with it on ovulation. Indeed the first-born of these clone mice has been named Cumulina.

Photo: Three generations of cloned mice. Top:  Nucleus donor. Middle and bottom rows:  second and third generations of cloned mice, respectively.

"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. Ryuzo Yanagimachi, professor in the Department of Anatomy and Reproductive Biology at the John A. Burns School of Medicine of the University of Hawaii.

The mice were produced with a new method called the Honolulu technique, developed by Dr. Teruhiko Wakayama, also at the University of Hawaii. 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 genetically identical mice.

"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 success of this project followed years of failed attempts at cloning mice by nuclear transfer. The Honolulu team also tried nuclei from nerve cells and Sertoli cells from the testis, which are also permanently in the G0 stage. But only the cumulus cell nuclei were successful.

The clone offspring appear to be normal and have already produced offspring of their own in the 'natural'  way. The success rate of the cloning technique ranges from 1 in 40 to 1 in 80 survivors for every embryo implanted.

The new method requires split-second timing. Within five minutes of the donor nucleus removal, the researchers insert it into the developing egg cell or 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."

The latest cloning experiment promises to provide both scientific and commercial rewards. The cloning of mice should help enhance understanding of technical and biological factors that contribute to successful cloning. Developmental biologists will also be able to address the crucial question of how the donor nucleus from a specialized cell becomes reprogrammed by the egg cytoplasm to enable it once again to give rise to all the different cell types in the animal body. These studies in turn could improve understanding of the cellular and molecular componenets of aging and diseases such as cancer, AIDS, diabetes and multiple sclerosis.

On the commercial side, the Honolulu technique, could prove more useful for the production of drugs using transgenic animals than earlier techniques because of its efficiency of reproducibility. The researchers believe the same technique can be applied to larger animals. This may make it easier to produce transgenic animals capable of producing therapeutic human proteins such as insulin and clotting factors.

There are also plenty of potential uses for cloned mice. 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. We really know very little about the mechanism of early development, and this cloning technique should help us to learn much more." noted Dr. Anthony Perry, also at the University of  Hawaii.

"Research in mice is much less expensive than in large animals such as sheep and cattle and the work published today will encourage many laboratories -- including our own -- to use mice in studies to understand the basic mechanisms involved in the 'reprogramming' of adult cells.  Studies on mice will also facilitate the use of nuclear transfer for studying mechanisms involved in aging and in cancer," said Dr. Ian Wilmut, the Roslin Institute researcher who brought Dolly the sheep clone into the world.

The study appears in the July 23, 1998 issue of Nature.

Related information on the Internet
 Cloning News and Resources
 Hello Dolly: Sheep Clone
 Graphic: the Honolulu Technique

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