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Researchers offer proof-of-concept for Altered Nuclear Transfer

Scientists at Whitehead Institute for Biomedical Research have successfully demonstrated that a theoretical--and controversial--technique for generating embryonic stem cells is indeed possible, at least in mice.

The theory, called altered nuclear transfer (ANT), proposes that researchers first create genetically altered embryos that are unable to implant in a uterus, and then extract stem cells from these embryos. Because the embryos cannot implant, they are by definition not "potential" human lives. Some suggest that this would quell the protests of critics who claim that embryonic stem cell research necessitates the destruction of human life. Scientists and ethicists have debated the merits of this approach, but so far it has not been achieved.

"The purpose of our study was to provide a scientific basis for the ethical debate," says Whitehead Member Rudolf Jaenisch, lead author on the paper that will be published in the October 16 online edition of the journal Nature. "Our work is the first proof-of-principle study to show that altered nuclear transfer not only works but is extremely efficient."

First proposed by William Hurlbut, Stanford University professor and member of the President's Council on Bioethics, ANT has been described as an ethical alternative to somatic cell nuclear transfer (SCNT), also known as therapeutic cloning.

For SCNT, a donor nucleus, for example one taken from a skin cell, is implanted into a donor egg cell from which the nucleus had been removed. This egg cell is then tricked into thinking it has been fertilized. That causes it to grow into a blastocyst--a mass of about 100 cells--from which stem cells are removed. These embryonic stem cells can divide and replicate themselves indefinitely, and they can also form any type of tissue in the human body. However, to cull these stem cells, the blastocyst must be destroyed, which some critics insist is tantamount to destroying a human life.

The proce dure theorized by Hurlbut is similar to SCNT, but with one crucial twist: Before the donor nucleus is transferred into the egg cell, its DNA is altered so that the resulting blastocyst has no chance of ever becoming a viable embryo. As a result, a "potential human being" is not destroyed once stem cells have been extracted.

Jaenisch--a firm supporter of all forms of human embryonic stem cell research--has shown that technical concerns about this approach can be overcome.

Jaenisch and Alexander Meissner, a graduate student in his lab, focused on a gene called Cdx2, which enables an embryo to grow a placenta. In order to create a blastocyst that cannot implant in a uterus, the researchers disabled Cdx2 in mouse cells.

They accomplished this with a technique called RNA interference, or RNAi. Here, short interfering RNA (siRNA) molecules are designed to target an individual gene and disrupt its ability to produce protein. In effect, the gene is shut off. Jaenisch and Meissner designed a particular form of siRNA that shut off this gene in the donor nucleus and then incorporated itself into all the cells comprising the blastocyst. As a result, all of the resulting mouse blastocysts were incapable of implantation.

However, once the stem cells had been extracted from the blastocysts, Cdx2 was still disabled in each of these new cells, something that needed to be repaired in order for these cells to be useful. To correct this, Meissner deleted the siRNA molecule by transferring a plasmid into each cell. (A plasmid is a unit of DNA that can replicate in a cell apart from the nucleus. Plasmids are usually found in bacteria, and they are a staple for recombinant DNA techniques.) The stem cells resulting from this procedure proved to be just as robust and versatile as stem cells procured in the more traditional fashion.

"The success of this procedure in no way precludes the need to pursue all forms of human embryonic stem cell resea rch," says Jaenisch, who is also a professor of biology at MIT. "Human embryonic stem cells are extraordinarily complicated. If we are ever to realize their therapeutic potential, we must use all known tools and techniques in order to explore the mechanisms that give these cells such startling characteristics."

ANT, Jaenisch emphasizes, is a modification, but not an alternative, to nuclear transfer, since the approach requires additional manipulations of the donor cells. He hopes that this modification may help resolve some of the issues surrounding work with embryonic stem cells and allow federal funding.


Source:Whitehead Institute for Biomedical Research

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