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The Clone Reimagined

only 31 of every 100 human conceptions complete the journey.

By | April 25, 2005

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Normal development is an inefficient process – only 31 of every 100 human conceptions complete the journey. Development via somatic cell nuclear transfer (SCNT), under the direction of adult DNA from a specialized cell is even less efficient, with success rates of about one in 300 or worse. Although nearly as many questions remain as when SCNT was first attempted half a century ago, researchers are closing in on why it is so difficult to emulate normal development starting with this most unusual genetic headquarters.

The major distinction between development via sperm and egg versus SCNT is a profound perturbation of gene expression. The deviations arise from various sources, including influences of the culture medium and factors in the nucleoplasm and cytoplasm. When all goes well, morphogens interact to effectively reprogram the new nucleus, which is vital to resetting the developmental program. But even the most prominent researchers readily admit that much of what transpires in SCNT is a mystery. Says John Gearhart, director of stem cell biology at the Institute for Cell Engineering at Johns Hopkins University, "If you figure out how to reprogram a nucleus, let me know."

MORE THAN NEW DNA

Biologists know some steps in the basic choreography of fertilization. In most mammals examined, just before the male and female pronuclei meet and merge, the paternal genome is globally and actively demethylated, and the maternal genome passively follows. About when the blastocyst implants in the uterine wall, the first inklings of determination and then differentiation begin as methylation ensues in a stepwise, lineage-specific manner. This epigenetic pattern is stamped on further cell generations. Imprinted genes escape the initial CH3 removal, remaining tenaciously methylated at key stretches of CpG dinucleotides. For these genes, only one parent's allele is normally expressed in offspring.

"The goal in nuclear transfer (NT) is to reproduce the conditions of a very early, one-cell embryo produced by fertilization, because these embryos clearly develop very nicely," says Tony Perry, head of the laboratory of mammalian molecular embryology at the Riken Center for Developmental Biology in Kobe, Japan. But SCNT is fraught with difficulties, both in technique and assumptions. Even the seemingly logical assumption that clones would be identical rarely holds up.

"Animal clones may have greater variability in some traits than conceiving the old-fashioned way," says Randy Jirtle, a professor of radiation oncology at Duke University Medical Center. Cloned cat CC, for example, differs from her nucleus donor in coat color due to different inactivation patterns at X-linked coat-color genes. Perhaps less anticipated were the distinctive personalities of the two cats (although cat owners might not be surprised – felines do things as they see fit, even accessing their genomes). And pigs cloned at Texas A&M University vary significantly in levels of certain blood components.1

FIFTY YEARS OF NUCLEAR TRANSFER

CC and the Texas porkers are members of a select club: the successfully cloned. In 1952, Robert Briggs and Thomas J. King first transferred nuclei from somatic cells to enucleated eggs and watched development unfold in the northern leopard frog Rana pipiens. In the 1960s and 1970s, SCNT experiments continued in the South African clawed frog, Xenopus laevis, by John Gurdon and others. Decades later Dolly the sheep, Cumulina the mouse, and cloned calves Charlie and George brought mammals into the society, demonstrating that SCNT can succeed – albeit rarely and problematically.

"Briggs, King, and Gurdon were an excellent starting point for a student of nuclear transfer. The problem is that they are also something of a finishing point," says Perry. Little has been learned since then about what happens when SCNT works. What is known, generally, is that nuclear reprogramming is the first major hurdle a cell created by SCNT must surmount. In successful cloning experiments, the problem appears to work itself out, although whether development unfolds normally isn't known. A relocated somatic cell's genome must silence those genes that made it part of a breast or connective tissue, while activating the genetic program that normally runs in a fertilized ovum.

The basic problem: A somatic cell's DNA in an egg is a decidedly unnatural circumstance. Eclectic experiments are revealing some of the points at which the unusual embryonic journey of SCNT goes awry.

CULTURE SHOCK AND TRANSFER WOES

Culture medium that nurtures donor cells can affect later gene expression if it contains nutrients that methylate DNA. This connection became evident rather unexpectedly when assisted reproductive technology registries began reporting cases of extremely rare human imprinting disorders, such as Beckwith-Wiedemann syndrome, Angelman syndrome, and retinoblastoma, associated with in vitro fertilization (IVF).2 Although the higher incidence could reflect reporting bias or underlying infertility issues, another explanation is that the culture medium methylates certain imprinted genes in cells destined for IVF. Jirtle's group has shown that single carbons from methionine and choline form the basis of methyl groups, and folic acid, vitamin B12, and pyridoxal phosphate are cofactors essential for methyl metabolism.3

If methionine in culture medium causes imprinting disorders in children born after IVF, it might also alter methylation of DNA destined for SCNT. "We must learn how to optimize the media to maintain epigenetic profiles," says Jirtle.

<p>THESE LITTLE PIGGIES:</p>

Courtesy Jorge Piedrahita

The cloned pigs show similar, yet distinct spotting patterns due to epigenetic influences. The migration of pigmented cells is affected by random phenomena.

Rudolf Jaenisch, professor of biology at the Massachusetts Institute of Technology, and colleagues investigated an effect of media conditions by comparing expression of 10,000 genes in the livers and placentas of mice cloned from "stale" cultured embryonic stem cell (ESC) nuclei or "fresh" cumulus cell nuclei.4 All the mice had large offspring syndrome (LOS), which affects most cloned mammals as well as those derived from assisted reproductive technologies. The researchers found double the normal expression for 286 genes from cumulus-cell clones and 221 genes from ESC clones, with 76% overlap. The affected genes encode proteins essential for embryogenesis, including growth factors, receptors, enzymes, clotting factors, MHC antigens, and histone deacetylases. But determining whether or not these gene-expression profiles were the result of culture conditions required further experiments.

Using a manipulation called tetraploid complementation5 they isolated an effect of culture conditions. This technique overcomes a major limitation of cloning from ESCs – they cannot create a placenta. Normally, explains Jaenisch, ESCs are injected into a diploid blastocyst, and the resulting mosaics must be bred to obtain a clonal animal.

Using tetraploid helpers generates ESC-derived embryos faster, Jaenisch says. "Two cells fuse to form a tetraploid cell that divides to form a very nice blastocyst, but it doesn't develop. The tetraploid cells exclude the epiblast lineage, so they don't make an embryo." But mix embryonic stem cells and tetraploid cells, "and we get a mouse embryo that is entirely derived from ES cells, but supported by a tetraploid placenta." Such an embryo is a clone of the embryonic stem cell, but it is not necessarily derived using SCNT, Perry points out.

Unlike NT-derived mice using ESC or cumulus cell nuclei, the mice whose development began with the help of tetraploid extraembryonic structures had normal placentas and no LOS. Expression of some genes, however, differed in the NT ESC-derived mice and the tetraploid-derived mice, revealing effects on the ESC clones caused by NT.

But the observation that the expression of some genes was altered in embryos derived from the two sources of ESCs (NT and tetraploid complementation), but not in cumulus-derived embryos, suggests that some of the differences in gene expression in a clone reflect nucleus source.

THE CENTER OF IT ALL

<p>IMPRINTED:</p>

© 2003 John Wiley & Sons

Just after fertilization, a global demethylation event occurs in the zygote, first in the paternal pronucleus, followed by the maternal pronucleus. Imprinting established during gametogenesis must resist this demethylation process. Remethylation of the diploid genome occurs post-implantation and sets secondary imprints that are maintained for the life of the individual. (From S.K. Murphy, R.L. Jirtle, BioEssays, 25: 577–88, 2003.)

The amphibian work of the 1950s and 1960s revealed that the younger or less specialized the donor nucleus, the higher the success rate of SCNT. Results in mice are similar: Transferring nuclei from 2-celled embryos leads to a 22% birth rate, yet that number plummets to 14% for a 4-cell-stage donor and to 8% for an 8-cell-stage donor.6 Only 1–3% of nuclei transferred from differentiated cells from adult frogs support full prenatal development. Using donor cells functionally related to the germline increases those odds slightly.

But even the most specialized cell does not have its genetic headquarters irreversibly silenced. Groups at Harvard University and Rockefeller University cloned mice from highly specialized olfactory sensory neurons (OSN).78 Each OSN expresses only one of roughly 1,500 olfactory receptor genes, but clones had the full olfactory repertoire indicating donor-nucleus reprogramming.

The list of adult cell types whose nuclei can support at least limited development is growing. Jaenisch and Konrad Hochedlinger had previously cloned mice using a distinctive adult somatic cell nucleus donor – B cells, which normally rearrange their genomes in response to exposure to antigen. Every cell in the cloned mice had the altered genome characteristic of the donor B cell.9 Even cancer-cell nuclei, in mice, can recapitulate very early development. Jaenisch's group used DNA from leukemia, lymphoma, and breast cancer cells to generate preimplantation embryos,10 and James Morgan's group at St. Jude Children's Research Hospital in Memphis, Tenn., used nuclei from medulloblastoma cells.11

If SCNT works with a donor cell so specialized it is dubbed "terminally differentiated" – as with an OSN – then anything is possible, given the appropriate signals. And that's where the nucleoplasm and ooplasm enter the picture.

JUMPSTARTING THE PROGRAM

<p>GLOWING GOOD RESULTS:</p>

© 2004 Nature Publishing Group

A mouse derived from a mature olfactory sensory neuron nucleus contained the complete olfactory repertoire. This mouse was created using tetraploid complementation from an OSN3 embryonic stem cell line expressing green fluorescent protein. (From K. Eggan et al., Nature, 428:44–9, 2004.)

Once a somatic cell nucleus is transferred, factors in the recipient ooplasm stimulate reprogramming. At this point, the egg cells – technically still oocytes before fertilization – have had their DNA removed and are arrested at metaphase of the second meiotic division, when the nuclear membrane temporarily breaks down. "Metaphase II chromosomes are removed and not nuclei. The question is if and which crucial nuclear proteins are left behind," says Hans Schöler, director of the Max Planck Institute for Molecular Biomedicine in Munster, Germany.

Schöler and Jeong Tae Do, both formerly at the Center for Animal Transgenesis and Germ Cell Research at the University of Pennsylvania, demonstrated a role for nucleoplasm by fusing ESCs and their derivatives with neurosphere cells (NSCs), as a model of induced dedifferentiation that is easier to work with than oocytes.12 Fusing an NSC with an entire ESC activates Oct4, a gene expressed in pluripotent cells of the embryo up until gastrulation, which is not normally expressed in NSCs, and therefore marks reacquired pluripotency. The researchers then separated ESCs into karyoplasts (membrane-bounded nuclei with a hint of cytoplasm) and cytoplasts (membrane-bounded cytoplasm). NSCs fused with ESC karyoplasts turned on Oct4, but NSCs fused with ESC cytoplasts did not. Therefore, the ESC nucleus contains factors necessary for reprogramming. Further, when the researchers blocked DNA replication by treating the ESC karyoplasts with mitomycin C, Oct4 was still expressed – indicating that something in the nucleus other than DNA plays a role in reprogramming.

Just as important as turning on genes such as Oct4 is turning off the genes that sculpted characteristics of the donor cell. John Gurdon and Ray Ng at the University of Cambridge showed that cells of cloned Xenopus "remember" their origin and express genes normally active in the nucleus donor cell.13 When the researchers transferred nuclei from neuroectoderm into enucleated oocytes, some of the resulting clones not only overexpressed the neuroectoderm marker Sox2, but did so in their endoderm, indicating that the inappropriate gene expression was both out of time and out of place.

Skewed expression of Oct4 and Sox2 are but two ways that SCNT-derived offspring veer from normalcy. If aberrant gene expression ensues and persists whenever a somatic cell nucleus finds itself in ooplasm – as Jaenisch's 10,000-gene arrays suggest – it isn't surprising that SCNT leading to birth is exceedingly rare.

Taken together, experiments that correlate unusual gene expression patterns with the events of NT are revealing many points at which development leaves its normal path – suggesting that a clone such as Dolly may have superficially been just a sheep, but her tissues may have told a different story. It is perhaps ironic that as politicians and the public continue to debate SCNT in tissue engineering, and to universally condemn its use for human reproductive cloning, developmental biologists are discovering that in reproduction, the ends do not necessarily reflect the means.

Kevin Eggan, a junior fellow in the department of molecular and cellular biology at Harvard University, who was involved in cloning a mouse from an olfactory neuron last year, sums up, "Everything we know from animal experiments suggests that a cloned child would likely be in danger of a number of developmental abnormalities, both known and unknown."

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