[Note: Nature Genetics has embargoed this article until May 27, 2002 2 p.m. EST]
New breakthrough research reported by scientists at the University of Connecticut in today's issue of the journal Nature Genetics, suggests that abnormal expressions of genes in the organs of cloned cows may explain why so many cloned pregnancies fail and why some cloned animals suffer medical problems and death after birth.
The scientists examined the expression patterns of 10 genes on the X chromosomes in 9 full-term clones (4 alive and five dead) and 1 aborted fetus cloned from one of the clones. All these clones were derived by nuclear transfer using different somatic cells of an aged Holstein cow. They found abnormal gene expressions in the major organs including heart, liver, brain, kidney and spleen of all dead clones while live clones appear normal in gene expressions in their blood and skin tissues.
The research focusing on how cells interpret their genetic instructions sheds new light on why cloning by somatic cell nuclear transfer suffers from low success rates. The findings also raise questions how cloned cows can develop to full term despite the problems of abnormal gene expression in their organs.
"Our study demonstrates that in clones, even though they can develop to full term, many abnormalities in gene expression exist which may be partially responsible for the developmental abnormalities frequently observed, including death," says Dr. Xiangzhong (Jerry) Yang, lead author of the article and Director of UConn's Center for Regenerative Biology.
Dr. Thomas Wagner, a Clemson University professor of molecular biology, said the UConn study demonstrates that cloning is a maturing technology. "Dr. Yang's study clearly shows that cloning, like any biotechnology, needs to be perfected," said Wagner. "His results identify a reason why it doesn't always work and offers a means to make modifications in the procedure to correct the problem."
Numerous cloned mice, cows, goats and pigs have been born since Scottish scientists cloned the first sheep, Dolly, in 1997. Yet it takes dozens or sometimes hundreds of attempts to succeed. Only few clones are long-term survivors as 80 percent die during pregnancy or soon after birth from defects suggesting that during embryonic and fetal development the genes did not work properly.
The "gene expression" investigated by Yang's new study is a process in which cells use only the parts of the genetic codes to accomplish their special function. So while different kinds of cells such as skin, muscles, and nerves all contain the entire DNA instruction book for creating another cow, say, the only part of their DNA that becomes active is that which turns on genes, which determine the kind of the cells they become.
Once a cell has taken on its specialized role, many of the other genes in the cell become inactive. The wonder of cloning is that when DNA of any single cell is transferred into an empty (no DNA) donor egg cell, the inactive genes in the donor nucleus are quickly reprogrammed and become once again able to set off development of a new cow.
According to Yang's study, abnormalities seen in cloned cattle may stem from faulty or incomplete "reprogramming" of a cell's nucleus. During this crucial step, after researchers transfer the DNA from a differentiated cell into an egg cell that has been emptied of its own DNA and the reconstructed egg cell commences to reset the gene expression of the injected DNA, something goes awry.
For clones to commence development, genes normally expressed when cells are united during fertilization, but silent in the DNA of the donor cell, must be reactivated. In natural reproduction the early female embryos silence or inactivate nearly all genes in one of the two X chromosomes by the process of X chromosome inactivation, short as XCI. This is because the female embryos has two active X chromosomes, one too many compared to the male embryos. Mother nature evolved this dosage composition mechanism so that male and female would be equal in having expressed genes on their chromosomes.
In the cloning process, however, cloned female embryos receive a donor cell, which contained one active X and one inactive X chromosome. This is different from the situation in natural reproduction in which the female embryos start with two active X chromosomes and later silence one of them. In the study announced today, UConn scientists investigated whether and how the normal patterns of X chromosome inactivation are erased and re-established after cloning.
They compared 10 X-linked genes in various organs/tissues of nine cow clones (4 live and 5 dead clones) to control cows born from natural reproduction. "We found all 10 genes examined were expressed in the organs of the normal control cows," said Yang. "In contrast, we found abnormal expression patterns in nine out of the ten X-linked genes in major organs of deceased clones.￣
"In addition, the degree of abnormal expression profile of X-linked genes in a particular organ varied from clone to clone and in individual clones from organ to organ," Yang continued. ＾These perhaps explain why cloned animals manifest different forms of abnormalities and die at different stages of gestation. Yang added. "Interestingly, we did not find any abnormal expression of these X-linked genes in blood and skin from the live clones."
They also examined XCI in the placenta of clones. In natural reproduction, only the maternal X chromosome is active in the placenta while the paternal X is inactive. Interestingly, they found that both X chromosomes are active in the placenta of the dead clones. ＾This finding may help to explain the commonly observed abnormal placental problems for cloned animals￣, Yang commented. However, in the placenta of the live clones, only one X chromosome is active. This suggests that the cloned embryo did not preferentially inactivate the paternal X chromosome, but may have maintained the XCI pattern of the donor cell in the placenta.
An earlier study at the Massachusetts Institute of Technology found that cloned mice did not experience reprogramming problems on X-chromosome reprogramming. This discrepancy may be explained by differences between species, said Yang. Additionally, in the mouse study whole mid-gestation fetuses were studied which may mask any aberrant expression patterns of X-linked genes if they existed in individual tissues and/or organs, he said.
UCONN scientists also found that different types of donor cells could affect cloning efficiency and clone viability. They attempted to clone cows from three different types of cells: ovarian cumulus, skin fibroblast and mammary epithelial. Four of the six calves from ovarian cumulus cells lived. By contrast, all four calves from skin fibroblast cells died within 24 hours and no calves were born from mammary epithelial cells.
"Our findings suggest that the incomplete reprogramming of these X-linked genes in the deceased clones may have contributed to the deaths of these clones," Yang said. " These results have obvious implications for human and animal cloning, and provide further warning that we still do not know enough about animal cloning to proceed without caution. Further understanding of the abnormal gene expressions in clones may help developing better cloning protocols to develop healthy clones￣.
For more information contact:
Dr. Xiangzhong (Jerry) Yang, Professor of the Department of Animal Science and Director of the Connecticut Center for Regenerative Biology
Tel: 860-486-2406
E-mail: jyang@canr.cag.uconn.edu
Website: (includes photos): http://www.oocities.org/uconnyanglab/
Additional comments may be requested from the following external experts:
Dr. Cindy Tian
Assistant Professor, Department of Animal Science/Center for Regenerative Biology; University of Connecticut
Tel: 860-486-9087
E-mail: xtian@canr.cag.uconn.edu
Dr. Robert H. Foote
Professor Emeritus, Department of Animal Science; Cornell University, Ithaca, NY 14853
Tel: 607‑255‑2866
E‑mail: rhf4@cornell.edu
Dr. Thomas Wagner
Professor of Molecular Biology; Director of Oncology Research, Greenville Hospital System,
Clemson University; Greenville, SC 29605‑5601
Tel: 864‑455‑1565
E‑mail: twagner@ghs.org
Assistant Professor of Biology; Centro de Estudos do Genoma Humano, Departamento de Biologia, Instituto de Bioci┷ncias, Universidade de São Paulo, São Paulo, Brazil
Tel: 011-55-11-3818-7476
E-mail: lpereira@usp.br
Professor of Immunogenetics; Director of the W. M. Keck Center for Comparative and Functional Genomics; University of Illinois/Champaign-Urbana; Urbana, IL 61801
Tel: (217) 333-5998
E-mail: h-lewin@ux1.cso.uiuc.edu
Assistant Editor
Tel: 212-726-9289
E-mail: m.stebbins@natureny.com
Manager of Media Relations, Office of University Communications, University of Connecticut
Tel: 860-486-5627
E-mail: david.bauman@uconn.edu
Index|Current News |Previous News |Nature Genetics Paper|PNAS Paper|Backgound|Related|Photos