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News Release
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For Immediate Release:
November 21, 2005
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Contact:
David Bauman, Office of University Communications, University of Connecticut, Tel: 860-486-5627; E-mail: david.bauman@uconn.edu
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Somatic cell reprogramming by nuclear transfer to totipotent embryonic stage is norm, similar to naturally fertilized embryos
Storrs, CT. – Cloned embryos can successfully reprogram themselves to resemble the equivalent of a nucleus of a naturally fertilized embryo, according to new collaboration study from University of Connecticut, University of Illinois and INRA, France.
The research sheds new light on how an adult somatic cell nucleus that has been transferred into an egg that’s had its nucleus removed, undergoes global gene reprogramming in a process called differentiation from a differentiated somatic cell genomic to an undifferentiated embryonic status.
The study, funded by a U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service National Research Institute Grant, appears in the December 6, 2005 issue of the Proceedings of the National Academy of Sciences.
“For the first time we have examined and compared gene expression profiles of individual cloned embryos to those of their nuclear donor somatic cells used for nuclear transfer, and then to those of naturally fertilized control embryos,” said Dr. Xiangzhong “Jerry” Yang, director of UConn’s Center for Regenerative Biology. “Our results showed convincingly that the cloned embryos have undergone significant nuclear reprogramming by the blastocyst stage.”
Since Dolly the sheep was first born in 1997, animal cloning using nuclei from adult cells, or somatic cell transfer, has been accomplished for at least 13 mammal species. However, this is a highly inefficient process: although the efficiency for embryo production at blastocyst stage via cloning is as high as or even higher than the routine in vitro fertilization technology, only a handful (1-5%), at most, of these blastocysts produces live young when implanted into a female, regardless of species.
One current hypothesis for this problem is that the adult nucleus from the somatic cell is not fully reprogrammed back to an embryonic state. Dr. Yang and his research team explored this problem by looking at the genes that are turned on and turned off in blastocysts produced through several mechanisms, including somatic cell nuclear transfer.
Their question was whether cloned embryos formed from somatic cells have normal or abnormal gene expression compared to naturally fertilized control embryos. And, if gene expression was abnormal, could this be part of the explanation for low cloning success rates.
The somatic cell nucleus that has been transferred into an egg undergoes reprogramming by the egg’s cytoplasm. This reprogramming process takes an adult cell nucleus and makes it into the equivalent of a nucleus of a new embryo, a process called dedifferentiation. This results in the formation of cells that are capable of making every type of cell in the body—totipotent cells—from the nucleus of a cell that had originally been differentiated into a specific cell type.
Using a technique called cDNA microarray technology, the UConn, UIUC and INRA research team looked at gene expression – which genes were turned off and which were turned on – in cattle nuclear transfer (NT) embryos. They compared this to gene expression in cattle embryos produced by in vitro fertilization (IVF), where a bull sperm fertilizes a cow egg, and in cattle embryos produced by natural fertilization via artificial insemination (AI).
Surprisingly, they found that one week after cloning the profiles of the NT embryos were drastically different from their donor cells and were more similar to those of AI embryos than IVF embryos. Less than 1% of the 5,000 genes analyzed differed more than 2-fold between NT and AI embryos, roughly the same as differences between genetically unrelated AI embryos.
This means that the nucleus went through reprogramming. In fact, the genes that were more heavily expressed in the NT embryos affected the cells’ mitochondria, carrier molecule activity, transporter molecule activity, RNA splicing and ion transporter activity. These are all activities and areas that would be expected to be highly active in a young, growing and maturing cell.
These findings indicate that cloned embryos undergo significant nuclear reprogramming at embryonic blastocyst stage after cloning, and the problems associated with cloning may occur during later embryo development, said Dr. Yang. “These findings are consistent with the recent findings that cloned embryos can be produced efficiently for early development and for generating embryonic stem cells lines in mice, cattle and humans. Additional work remains to further understand whether the aberrant and poor developmental competence of cloned embryos is caused by aberrant reprogramming – in gene expressions – during later stages of development such as re-differentiation of nuclear donor genome for tissue and organogenesis, and whether small reprogramming errors during early development are magnified downstream in later development,” he said.
For more information contact:
Dr. Xiangzhong (Jerry) Yang, Professor and Director of Center for Regenerative Biology, University of Connecticut
Tel: 860-486-8728
E-mail: xiangzhong.yang@uconn.edu
Website: http://web.uconn.edu/crb
Additional comments may be requested from the following experts:
Dr. Cindy Tian
Assistant Professor, Department of Animal Science/Center for Regenerative Biology; University of Connecticut
Tel: 860-486-9087
E-mail: xiuchun.tian@uconn.edu
Dr. Harris A. Lewin
Professor of Immunogenetics; Director of the Institute for Genomic Biology, University of Illinois Urbana-Champaign; Urbana, IL 61801
Tel: (217) 244-3404
E-mail: h-lewin@uiuc.edu
Dr. Jean-Paul Renard
Institut National de la Recherche Agronomique INRA; Uniyr De Biologie Du Development
Jouy-en-Josas, 92170 France
Tel: 33-1-34652594
E-mail: renard@jouy.inra.fr
List of suggested other experts for comments
Atsuo Ogura, Ph.D., D.V.M.
RIKEN Bioresource Center
3-1-1 Koyadai, Tsukuba-shi,
Ibaraki 305-0074, Japan
Tel: +81(0)298-36-9165
Fax: +81(0)298-36-9172
e-mail: ogura@rtc.riken.go.jp
Thomas E. Wagner, Ph.D.
Distinguished Professor of Molecular Biology
Director of Oncology Research
Greenville Hospital System
Clemson University
701 Grove Road
Greenville, SC 29605-5601
Tel: 864-455-1565
Fax: 864-455-1567
e-mail: twagner@clemson.edu
Charles Jennings, Ph.D.
Executive Director
Harvard Stem Cell Institute
42 Church St.
Cambridge, MA 02138
Tel: 617-496-4050
Fax: 617-496-6625
Email: charles_jennings@harvard.edu
Dr. Jose Cibelli
Distinguished Professor of Animal Biotechnology
Cellular Reprogramming Laboratory
Animal Science - Physiology
Michigan State University
1230 Anthony Hall
East Lansing, MI 48824-1225
Phone: 517 432 9206
Fax: 517-353-1699
"Jose Cibelli" cibelli@msu.edu
Eckard Wolf, Ph.D.
University of Munich
Institut für molekulare Tierzucht und Biotechnologie
Feodor-Lynen-Str. 25
81377 München
Tel: +49-2180-6800
Fax: +49-89-2180-6849
e-mail: ewolf@lmb.uni-muenchen.de
Questions and answers
1. What is the main finding from your research?
Our study was aimed at examining nuclear reprogramming in somatic cell cloned bovine embryos utilizing cDNA microarray technology. The current hypothesis for the low efficiency observed in somatic cell nuclear transfer is that the donor cell nucleus is incompletely or aberrantly reprogrammed. For the first time, we have examined and compared the global gene expression profiles of single cloned embryos to those of the nuclear donor somatic cells used for nuclear transfer and to those of naturally fertilized control embryos. We found that the gene expression profiles of the cloned embryos were vastly different from those of their nuclear donor cells, demonstrating significant nuclear reprogramming of the somatic donor cell nuclei after nuclear transfer. Additionally, we also found for the first time, that the global gene expression profiles of the cloned embryos closely resembled those of the naturally fertilized control embryos, more so than embryos produced by in vitro fertilization. This suggests a relatively complete and efficient nuclear reprogramming of the differentiated donor nucleus to a developmentally totipotent status. Our findings in this study comparing cloned embryos with their somatic nuclear donor cells and with naturally fertilized control embryos revealed for the first time that by the blastocyst stage, the cloned embryos have undergone significant nuclear reprogramming.
2. Doesn't your finding contradict what most people assumed was the problem with cloning?
Yes, see #1.
3. So if things are not going wrong at the genetic level, what do you think is causing the pregnancy success rate to be so low?
This positive evidence indicates that the low developmental efficiency and dramatic pregnancy loss following nuclear transfer may not be caused by the early nuclear reprogramming during embryo development, but potentially caused by reprogramming problems during postimplantation fetal development. Thus, the results from our study represent a substantial advance in the understanding of nuclear reprogramming and suggest a two stage process: 1) de-differentiation after nuclear transfer during preimplantation development and 2) re-differentiation during organ and tissue-genesis during postimplantation fetal development.
4. ....and finally, what do you make of the finding that cloned embryos are less "screwed up" than IVF embryos? Is this a surprise too?
Very good comments. Our data is very comprehensive and convincing. By comparing the cloned blastocysts to the nuclear donor cells and to the AI embryos, we demonstrate that the nuclear transfer procedure has at least “de-differentiated” the donor nucleus to a state in which it can drive development to the blastocyst stage. Yes, our findings for the comparisons of the cloned embryos to the naturally fertilized AI embryos versus comparisons of the IVF embryos to the naturally fertilized AI embryos are surprising. It is commonly known that IVF embryos have a calving rate of 35-40% while NT embryos have a calving rate of 5-10%; we expected that NT embryos would be more variable and more significantly different in global gene expression when compared with the naturally fertilized AI embryos. However, our findings to the contrary are surprising and interesting as the reviewer stated, “Interestingly, the authors also find that the gene expression profiles between cloned and AI embryos were more similar than those between the IVF and AI embryos.” Our observations and conclusions are convincing as the reviewer stated, “The developmental inefficiency of cloning is unlikely to be due to a failure of nuclear reprogramming during early embryo development.” It is expected that the comparisons between the IVF embryos and the naturally fertilized AI embryos as well as among individual IVF embryos show significantly different gene expression because “IVF embryos have a calving rate of 35-40%,” much lower than that of naturally fertilized AI embryos following embryo transfer (50-60%).
5. Finally finally, what does your research tell us about the safety of cloning, either animals or humans?
Our results suggest that reproductive cloning’s efficiency in all mammalian species so far are still very poor, but potential therapeutic cloning for generating totipotent embryonic stem cells using somatic cell nuclear transfer in mice, cattle and humans are with very high efficiency (the success rate is about 10-20 times higher than reproductive cloning for term development in the mouse model).
The commonly observed low developmental efficiency of cloned embryos may not be largely due to nuclear reprogramming during early embryo development (reprogramming of the somatic donor cell genome from a differentiated to a totipotent status – i.e., gene de-differentiation), but potentially caused by abnormal gene reprogramming during postimplantation fetal/placental development. However, additional work remains to determine whether small reprogramming errors (<1% of genes examined) at the early stage are magnified downstream in development. The success of somatic cell nuclear transfer may be dependant upon nuclear reprogramming of gene expression for de-differentiation of the donor somatic cell nuclei during early embryo development, and reprogramming of gene expression for re-differentiation of cloned embryos during organ and tissue-genesis in later development.
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