Chromatin Transfer Promises to Improve Survival, Efficiency in Cattle Cloning, Journal Reports.

Science Writers/Health/Medical Writers

WESTPORT, Conn.--(BUSINESS WIRE)--Oct. 28, 2003

A new approach to animal cloning may increase the survival of cloned cattle, thereby overcoming key problems encountered in the widely-used process of nuclear transfer cloning, according to a report in the online edition of Biology of Reproduction, a scientific journal ("Papers in Press" at www.biolreprod.com (DOI:10.1095/biolreprod.103.021220)). The report, by scientists of Aurox LLC, describes the scientific community's first successes using a novel chromatin transfer technology to clone cows. Cloning animals has been proposed as a possible means of manufacturing unlimited quantities of therapeutically useful proteins, including fully human antibodies, against a wide variety of disease targets.

Aurox' development of the chromatin transfer technology has been supported by Hematech LLC, which holds a license from Aurox to apply chromatin transfer for the production of cloned calves that produce human polyclonal antibodies for potential therapeutic and biodefense applications. Dr. James Robl, President and Chief Scientific Officer at Hematech and an Aurox study investigator commented, "The chromatin transfer technique has helped advance Hematech's success in developing our antibody production system. Our ability to reliably and efficiently produce cows that generate our human antibody product will be a great boon as we grow our commercial operations."

"This journal report demonstrates that cloned animals, such as calves, can be reliably produced via in vitro manipulation of nuclei afforded through chromatin transfer," said Philippe Collas, Ph.D., lead investigator of the Aurox study and Professor at the Institute of Medical Biochemistry, University of Oslo. "We may be able to determine if and how the chromatin is 'repackaged' when nuclei and cells are functionally reprogrammed and identify which molecules do the job. And importantly, by using chromatin transfer to create cloned embryos that more closely resemble normal embryos, we reported a trend towards lessening the number of tries needed to produce a viable pregnancy in cattle, and greater survival of the cloned calves at one month after birth."

Nuclear Transfer vs. Chromatin Transfer

In conventional nuclear transfer, the intact nucleus from an adult or somatic donor cell, such as a skin cell, is placed into an oocyte (egg cell) from which the genetic material -the chromosomes - has been removed. The oocyte is then activated and divides to become a cloned embryo. As a result of differentiation into their specific cell types, the nuclei of somatic cells exhibit a different pattern of markers compared to the nuclei of normal embryonic cells that have not yet differentiated themselves into specific cell types. These differences may lead to some of the widely reported limitations to the application of nuclear transfer in mammalian cloning, including low rates of embryonic development, high rates of pregnancy loss and low survival of cloned offspring.

The goal of the new chromatin transfer system is to produce a cloned embryo that more closely resembles a normal embryo. The first step is to create holes in the donor cell membrane (permeabilize) to provide direct access to the cell nucleus. Soaking the permeabilized cell in a special media causes the membrane of the nucleus to dissolve and enables removal of specific somatic cell regulatory proteins (remodeling) from the chromatin (genetic material and associated proteins). The permeabilized cell, containing the remodeled chromatin, is then fused to an egg cell and activated to create a cloned embryo.

About the Study

In the study, Aurox researchers examined the behavior of the donor nucleus in nuclear transplant embryos and identified several nuclear defects, including assembly of the differentiated cell-specific structural protein, lamin A, enhanced content of pronuclei in TBP, a general transcription factor, and high resistance of DNA to DNAse, all of which may affect development of the embryo. The researchers hypothesized that these abnormalities may result from incomplete remodeling of the donor cell nuclei and/or from mis-regulation of expression of differentiated cell-specific genes. Aurox researchers developed a system for reversibly permeabilizing the donor cell to allow direct remodeling of donor nuclei in vitro (i.e., in a test tube). Transplantation of the resulting permeablized cell, containing remodeled chromatin into recipient oocytes alleviates these defects and yields nuclei that more closely resemble the nuclei of normal embryos. The DNA in chromatin transfer embryos showed increased sensitivity to DNAse treatment, suggesting that it is in a more "open" configuration (like embryonic chromatin). In addition, the nuclear remodeling lowered TBP content in nuclei and induced repression of lamin A gene expression in the cloned embryos. Implications of repression of lamin A expression may include enhanced availability of factors required to turn on proper sets of embryonic genes and/or necessary to help turn off additional donor cell-specific genes. By permeabilizing the donor cell to allow condensation of the donor chromatin in vitro and inserting the condensed chromatin into the recipient oocyte, the researchers eliminated many DNA-bound components from the cloned embryos such as transcription factors or other potentially inhibitory somatic components. Finally, researchers used the chromatin transfer technique to create cloned bovine embryos and produced healthy offspring.

The research was supported by Hematech, LLC, the Norwegian Cancer Society and the Research Council of Norway and will be published in the January 2004 print edition of Biology of Reproduction. The research team was lead by Philippe Collas, Professor at the Institute of Medical Biochemistry, University of Oslo, and conducted in collaboration with Aurox researchers, Eddie J. Sullivan, Sriranjani Kasinathan, Poothappillai Kasinathan and James M. Robl.

About Aurox LLC

Aurox LLC is a technology development company currently focused on improving animal cloning and related technologies. The company is currently offering licenses worldwide to its novel technology, chromatin transfer, for animal cloning for agricultural, pharmaceutical and related purposes. For licensing opportunities, please contact James Barton, Chief Executive Officer of Aurox, at [phone].