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
"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.