by Leon CHAILAKHYAN, RAS Corresponding Member, and Tatyana SVIRIDOVA-CHAILAKHYAN, Cand. Sc. (Biol.), research scientist

The very possibility of human cloning has made quite a stir among the scientific community of many countries, Russia including, and broad public. It has triggered off a spate of lively discussions of all the various aspects of the problem. At the close of the century British, American and Japanese research scientists scored great achievements in the cloning of laboratory animals-farm animals for the most part. It all began with the sensational publication in the journal Nature (1997) about the cloning of the sheep Dolley. Yet a good deal of spadework preceded that momentous event - in this country too, in particular, at our Institute of Biological Physics of the Academy of Sciences of the USSR (today RAS Institute of Theoretical and Experimental Biophysics) which was involved in the job back in the 1980s.

Reconstruction of embryonic cells (that is replacing the native cell nucleus with a foreign one) allows to attack the all-important problem of identifying the mechanisms responsible for the realization of genetic information. Of much interest here are studies related to what is known as the phenomenon of totipotency ^* of a genome of differentiated cells - for one, to what extent the cell genome is amenable to reprogramming, and capable of producing paternal and maternal copies (cloning in other words), and also of giving rise to interspecies chimeric (or mosaic) animals. And so forth. Reconstruction of embryonic cells is also important for a global problem first raised by Professor B. Veprintsev of our Institute still in the 1970s - namely, pertaining to realization of the genetic information contained in the cryoconserved genomes of endangered animal species. But this is far from a complete list of cell reconstruction techniques.

Certain progress achieved in the cloning of amphibians during the 1960s and 1970s inspired confidence in the essential possibility of reprogramming other classes of vertebrates, mammals in particular, as an intriguing opportunity. But then we learned: transplanting the nuclei of mammalian embryonic cells is technically much more sophisticated. The point is that mammalian egg cells are far more sensitive to microsurgery, for their dimensions are thousands of times as small as those of the amphibians. Say, to extract the nucleus of a frog zygote (fertilized egg cell) or introduce it intact into an enucleated zygote, we need sharpened pipettes with the tip of no less than 10-20 mkm. But for cells 70-80 mkm in diameter (mouse zygotes) such pipettes are no good, for they destroy the substrate.

In a nutshell, research scientists involved in the cloning of mice or

^* Totipotency - differentiation capability of a genome represented by one cell introduced into an enucleated (devoid of a nucleus) egg cell of the same animal species thus inducing partial or complete embryonic and postembryonic growth of the organism.- Auth.

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other mammalians had to cope with what looked like a forbidding problem: how to transplant cell nuclei and keep the surface cell membrane intact?

This problem was solved with much success by Mac Gras and Salter who published their data in the journal Science in 1983. Their results became a landmark in conceptualizing the cloning of mammalians. They demonstrated that given certain experimental conditions (first and foremost, the presence of cytochalasin В in the incubation medium) and using micro-pipettes, it is possible to pipette off pronuclei ^* from a zygote together with part of the cytoplasm and surface membrane without damaging the zygote and obtain a caryoplast. ^**

Here's what we learned from our experiments: after the puncture of the zona pellucida (transparent germinal membrane) the micropipette enters the cell without damaging its surface membrane. This is largely due to the action of cytochalasin В which enhances the resistance of the cell to mechanical lesions in the process of enucleation by inducing decondensation of the cytoskeleton microfilaments and building up the plasticity and adaptivity of membrane surfaces to various mechanical effects. The caryoplast, as said above, is formed when pronuclei cum cytoplasm and surface membrane is drawn into a micropipette. Once both pronuclei are in, the pipette is carefully withdrawn from the zona pellucida. A cytoplasmic strand trailing after gives rise to two "cells" as it were or rather, forms two isolated compartments - the cytoplast (nucleus-free zygote) and the caryoplast, i.e. artificial cell division is induced thereby.

__ ^* Pronuclei - haploid (with one set of chromosomes) maternal and paternal nuclei. - Auth.

^** Caryoplast - pronuclei or diploid nuclei with adjacent cytoplasm that are completely isolated from the external medium by the surface plasma membrane. - Auth.

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So, the first step of genetic material transplantation from cell to cell is over: pronuclei are extracted from the zygote, while the surface membrane is intact. The second step is that of implanting the caryoplast into another cell (zygote) enucleated beforehand.

Attempts at injecting the caryoplast formed by pronuclei back into the zygote failed to come off: the caryoplast, always positioned outside the zygote, is in close contact with it. However, reconstruction of a new cell (zygote) from the cytoplast (enucleated zygote) and caryoplast is possible only if cell membranes fuse into one volume. For this purpose we decided to try an essentially new method - electric stimuli, or pulses (unlike Mac Gras and Salter who made use of the Sindai virus ^* for the fusion of the caryoplast and enucleated zygote).

At first we thought that the fusion of two neighboring cells by electric pulse should occur due to an irreversible electrical breakdown of two contacting membranes in their contact region. We searched for an optimal variant to have a voltage drop maximum across the contact region of the membranes.

We conducted a current between a big tungsten electrode outside and a tungsten filament within the micropipette. The pulse was generated at the moment when the caryoplast introduced under the zona pellucida came into contact with the enucleated zygote. Trying a wide range of currents, we failed to obtain the desired result nonetheless because of the significant electrophoretic transpositions of the medium within the micropipette, and that destroyed the caryoplast.

Then we modified our technique somewhat: one of the tungsten microelectrodes was to have its tip, 2-3 mkm in diameter, bare, while the other remained fully insulated as in the previous experiment. The first electrode fed pulses into the contact region. We could register occasional fusions of the caryoplast with the enucleated zygote. But as soon as the electrode was removed from the contact region, either the caryoplast or the cytoplast was destroyed (perhaps because of the high current density near the tip of the microelectrode it came to be heated and stuck to the substrate).

In a third variant of our experimental techniques we had the distance between the two electrodes several times as large as cell dimensions. In that case, mostly soon after the fusion, the reconstructed zygote perished (evidently the irreversible electrical break-down of the membrane extended beyond the contact region of the caryoplast and the enucleated zygote).

The most fortunate was an experiment with two electrodes - one had a 70-100 mkm tip, the other, 20-30 mkm, which was consistent with the dimensions of the enucleated zygote and caryoplast. The

^* Sindai virus - a pathogenic virus which, infiltrating membranes of adjacent cells, causes their fusion. - Auth.

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electrodes, except their tips, were well insulated. In that case the percentage of successful fusions rose to 75-90 percent.

This procedure was as follows: a prepared zygote and a caryoplast injected under the zona pellucida were placed tight between two electrodes, with the cytoplast/ caryoplast contact surface perpendicular to a straight line between the electrode tips. Then each pair of cells was stimulated by 20V electric pulses, each 0.1 ms long; the distance between the electrodes was ca. 100 mkm.

The caryoplast/cytoplast contact surface grew larger after electric stimulation. Subsequently the contacting membranes washed out or dissolved. The time between the electric stimulus and the evident signs of fusion varied from 1-15 to 20-30 min., i.e. by there orders. Yet the bulk of cells fused within 1 to 10 minutes. Additional pulses (two or three, at an interval of several seconds) were applied to the rest whereby the percentage of cell fusion increased appreciably.

To check on the viability of mouse zygotes reconstructed by electric fusion we transplanted them to foster females that produced progeny on time. These mice, both in growth and behavior, showed no differences from the common offspring of recipient mothers. The electric pulses used for the fusion in no way impaired the genome of a reconstructed zygote and can be employed in a wide range of cell engineering problems.

Our method has certain essential advantages over those used before. First, this is a universal technique - biologists know of cell types that cannot be joined together by means of polyethylenglycol or Sindai virus, while electrostimulation works without fail. Second, our method does not involve any chemical effects and contaminants noxious to cell viability. Third, it is convenient for experiment making, for one, by allowing a range of precise dosages. And last, our method makes it possible to fuse quite definite pairs of cells in targeted or selected fashion, something that cannot be done with the use of conventional techniques.

One important result of our research was that we obtained chimera mice through the combined use of microsurgery and electrically induced cell fusion.

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Mammalian chimeras are extensively used as a model system for studying various aspects of early embryonic growth and gene expression. Such organisms originate from two different cell populations whose contribution can be assessed both in quantitative and in qualitative terms with the aid of corresponding genetic markers; hence the immense interest in chimeras. Before our experiments there were two modes of obtaining chimeras: via aggregation of embryonic cells taken from animals of different lines and even species at the stage of cell division, and via injection of individual cells or their groups taken from one line into a blastocyst of another where they incorporated within the internal cell mass. But we applied an absolutely new method by replacing the nucleus of a two-cell mouse embryo with that taken from another line and combining microsurgery with electrofusion.

To introduce the donor cell nucleus into the enucleated blastomere we made use of targeted electrostimulation, with the contact surface of these two organelles being perpendicular to a straight line connecting the electrode tips. Positive results were obtained in 80 to 90 percent of the cases.

To evaluate the capacity of chimera embryos for growth, we transplanted them to foster female mice, and had a young chimera mouse born with a characteristic (mosaic) fur color.

Obtaining chimeras with the use of the above technique allows, in distinction from conventional methods, to follow the growth and distribution of embryonic material by means of blastomere markers, unambiguous and symmetrical with respect to genetic material.

Our optimistic forecast about a great future of the method (electrostimulated cell fusion combined with microsurgery) in cell engineering has come true. Late in the 1980s and in the early 1990s this very method was employed in Britain and the United States for the cloning of rabbits, cows and sheep. Used as donors were embryo cell nuclei in the stages of 8 to 64 blastomeres.

In the mid-1990s Dr. Willmut and coworkers in Britain made important headway in grappling with the problem of cloning. They cloned five sheep by taking as donor material cell nuclei from a culture of TNTU sheep embryo cells; the donor embryo was nine days old. The cell nuclei were subjected to as many as 25 passages in vitro. The donor nucleus and the recipient cytoplasm were synchronized according to the cell cycle. The zygote was reconstructed through microsurgery and electrostimulation. And finally, in 1997 the journal Naturecarried a sensational article on the cloning of the sheep Dolley that grew from a reconstructed zygote obtained via microsurgery and electrofusion; taken as donor was a cell

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nucleus from a mammary gland cell culture of a six-year-old sheep. This outstanding achievement, while opening up broad vistas for biotechnology, spawned many problems, as is often the case with pioneering discoveries. But was it possible to design other mammalian species?

One did not have to wait long for the answer. It came in July 1998 as Nature published an article proving that a reconstructed zygote with a donor somatic cell nucleus taken from an adult mouse could produce a real mouse. Then, in December 1998, four cloned calves were born, to be followed by yet another two cloned calves born in 1999 and 2000. The nuclei of the zygotes reconstructed in all these cases were taken from an aural cell culture of a 17-year old prize bull. In March 1999 American scientists cloned a calf from cells of a 21-year-old bull. And in January 2000 came the announcement about a cloned calf of a second generation, i.e. the nucleus of a zygote that gave rise to this individual was obtained from cells of a cloned bull. Finally, in March 2000 five hogs were cloned at Blacksburg, USA, using the same method as for Dolley. So the cloning of adult animals is a real thing for many mammalian species.

All too many question marks still remain. Here are the most important ones.

How viable is a cloned animal compared with the original? Doesn't its genome carry the load of events that have occurred with the original? This problem is today under active discussion relative to the dependence of the length of DNA telomere ^* sites and ageing. Dolley lived less than three years, but she gave birth to four lambs. How long will they live?

What cells of adult species are best for cloning? According to recent experiments of American scientists, a culture of fibroplasts ^** from adult animals is optimal for this purpose because the yield of transgene clones increases greatly thereby. That was the method used for the cloned sheep Polly with an active human gene.

What should be done to boost the efficiency of cloning to a two- digit figure percentagewise from mere fractions as it is today? How to make it productive for animal husbandry? To come on top of this problem we must find new approaches in preparing donor caryopiasts and recipient cytoplasts.

And last. The most intriguing and live issue: What about the cloning of man? Its prospects?

Human cloning is part and parcel of the general problem with all its implications, social and ethical for one. Yet its positive solution opens up wonderful opportunities to man in terms of medico- biological problem solving, in gerontology too. True, this may produce a breed of people with quite unusual characteristics and possibilities. And there are some other lurking dangers for the Homo sapiens too. So human cloning is a double-edged weapon. ^*** But be that as it may, it is impossible to check the progress of science, especially in areas touching the interests of many people. One should not impose a ban on research into this problem, but rather press for appropriate legislative and ethical enactments to keep it within legal bounds.

To conclude, we must call your attention to this aspect: besides the reproductive cloning of man (reproducing an adult man's genetic copy) and a host of socioethical problems that go with it, there is also another line of research - one related to medical and therapeutic cloning. It does not confront social or ethical problems, but can be used as a potent remedy against various pathologies. Say, 10-15-day human embryos grown in a Petri dish are used for obtaining stem cells which may become indispensable as cell substitute in cell therapy.

^* Telomeres - specific microscopic structures at each free end of the chromosome. - Auth.

^** Fibroblasts - cells of the connective tissue of animals and man . - Auth.

^*** See: A. Spirin, "Challenges of Contemporary Biology", Science in Russia, No. 2, 2001.- Ed.

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