news & views

nature genetics • volume 30 • march 2002 243

Mice cloned by somatic-cell nuclear trans-fer are born apparently normal in terms oftheir gross anatomy, behavior and theexpression of at least a subset—perhapsall—of their genes1,2. Their placentas areenlarged, however, and they can developadditional anomalies that include adultobesity and telomere elongation3–5. Onpage 253 of this issue, Narumi Ogonuki,Kimiko Inoue, Yoshie Yamamoto, AtsuoOgura and colleagues6 add hepatic andimmune impairment to this list. Like obe-sity, the new phenotypes show thatcloning can reveal or introduce errors thatdo not manifest themselves for months.The underlying mechanism may bethought of in terms of reprogramming,the process by which a donor nucleus thatwas one minute issuing instructions thatconstruct and maintain a given cell isminutes later issuing instructions to beginthe embryonic developmental programfollowing nuclear transfer. The presentdata contribute to an emergent picture inwhich the consequences of reprogram-ming can be subtle, specific and delayed.

These new cloning-associated patholo-gies and their cumulative effects need tobe described in greater detail, and expla-nations found for why the prematuredeaths occurred over a long time-frame(311 to 800 days, with some clones possi-bly not dying prematurely at all). If thereis a relationship between immune andhepatic phenotypes in cloning, it needs tobe established; it may, for example, reflectaberrations in a common stem cell popu-lation, or the hepatic lesions may havebeen a consequence of immune failure.Immune impairment has also beeninferred in cloned cattle7.

Cloning, from art to scienceTo what extent does cloning reveal anom-alies and to what extent does it introducethem? This question is addressed in theaccompanying figure. A potential illustra-tion of the issue is that whereas none of the

Untimely ends and new beginnings inmouse cloning

Anthony C.F. Perry1 & Teruhiko Wakayama1,2

1Advanced Cell Technology, One Innovation Drive, Worcester, Massachusetts 01605, USA. 2Center for Developmental Biology (CDB),2-2-3 Minatojima-minamimachi, Chuo-ku Kobe, 650-0047, Japan. e-mail: tony@advancedcell.com

A new study shows that the majority of mice cloned from somatic cells die prematurely. Of the six dead clones analyzed, four exhib-ited extensive necrosis of the liver, two had tumors and all had pneumonia, suggestive of immune impairment. These latest cloning-associated phenotypes will facilitate a better understanding of the mechanisms underlying nuclear-transfer cloning in mammals.

surrogate mother

relative contributions of oocyte anddonor nucleus to reprogramming are unknown

BOB CRIMI

donor strain influences cloning phenotype, but in what ways and whatis the mechanism?

are there cell-type–specific effects?

oocyte donor age is an unknown factor

influence of cultureconditions and cell cycle?

influence of cellularenvironment just prior to nuclear transfer

nuclear damage during collection; likely to vary even between workers using the same protocol

does cytochalasin havesubtle, pleiotropic effects?

oocytedonor

nucleusdonor

clonedmice

remove chromosomes in cytochalasin B

first microsurgicalprocedure on oocyte

long- and short-term effects of extensive microsurgery?

second microsurgicalprocedure on oocyte

majordeparture

fromfusion,which

lacks theinjection

step

nucleusmicroinjection

<0.5–10 hours

is chromosome/chromatindamage time-sensitive?

reprogramming is likely to occur duringthis time but little is known about how

oocyte activation

formation of 2–3pseudopronuclei

departure fromfusion, which

produces a single pseudo-

pronucleus embryo culture

embryo transfer

embryo culture conditions influencegene expression and development;is this significant?

donornucleus

collection

donorcellisolation

activation protocols are presumed to exertlittle influence on development, withoutgood evidence

The odyssey of the egg in mouse cloning. Schematic representation of the microinjection protocol used toclone mice1. Variables potentially influencing development are indicated in red.

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news & views

244 nature genetics • volume 30 • march 2002

clones described by Ogonuki et al.6 becameobese, around 20% of those previouslygenerated using the same strain (B6D2F1)and method did (ref. 5; T. Wakayama,unpublished data). One explanation is thatthe difference has essentially biologicalroots, reflecting the different nucleusdonor cell types used (cumulus cells previ-ously, Sertoli cells in the new study). Thedescription of somatic nucleus donorcell–specific cloning phenotypes woulditself be important. In this case, cloneswould represent an organismal read-out ofcell-type–specific mutation and epimuta-tion. Genetic heterogeneity in the nucleusdonor (for example, inbred versus hybrid)is already believed to influence develop-ment, including the obesity phenotype5,8.Collectively, these phenotypes may bemasked in offspring produced by nucleartransfer from embryonic stem (ES) cells;such clones have peculiar phenotypesreflecting the idiosyncratic nature of ES cells, and cloning by nuclear transferfrom ES cells is an anomalous paradigm9,10.

A second possibility is that variations inclone development are (partly) attribut-able to technical factors (see figure). Twoexperimentalists performing nucleartransfer side-by-side using materials fromthe same batch can produce irreconcilablydiffering data (unpublished observa-tions). It is noteworthy that few laborato-ries have reported mouse cloning fromadult somatic cells. The influence of tech-nique can be tested, but until operationalnuances are described and eliminated,they may produce cloning phenotypesattributed to other causes. Variance intechnical competence could also account,at least partially, for the range of efficaciesin other species such as cattle.

Less subtle technical considerations—such as the protocol used—might also con-tribute to cloning phenotypes. The

accompanying figure outlines the microin-jection procedure used by Ogonuki et al.6.In this method1, a nucleus is extracted witha pipette and injected into an enucleatedegg. The microinjection method is differentfrom the fusion method first used to clonesheep in 1986 (ref. 11) and later used to pro-duce Dolly. In that method the nucleus isintroduced by fusing the donor cell with anegg rather than by injection. It is possiblethat the methods are not equivalent in thedevelopmental outcome each produces;fusion presumably induces different sorts ofcellular trauma compared to microinjec-tion. So far, there is only a single compari-son of fusion versus microinjection inmouse cloning, and the numbers are toolow to be conclusive12. Additional parame-ters, such as the method of activation, mayalso exert a developmental influence.

Toward kinder, gentler cloningIn the context of the present study, it isthus possible that immune and/orhepatic impairment is a function not ofsomatic-cell nuclear transfer per se, butof the extensive micromanipulation itinvolved. This distinction is today an aca-demic one in that current technologiesnecessitate micromanipulation—the twoare inseparable. But it holds the promisethat one day, new nuclear transfermethodologies with less micromanipula-tion will generate offspring withoutcloning-associated phenotypes. Notably,Ogonuki et al.6 report that ‘controls’ gen-erated by micromanipulation (spermprecursor injection) were also susceptibleto greater premature death and tumori-genicity than the other control group,consistent with a phenotypic influenceby micromanipulation.

The awesome technical skills of theOgura laboratory have provided newinsights into cloning-associated pheno-

types and promise new departures in thestudy of reprogramming. Yet they saynothing about whether clones are subjectto altered cellular or organismal aging.This study does not, for example, reportclone telomere length, telomerase levels orother age-related parameters, althoughmice cloned in a previous study fromcumulus cells of the related strain,B6C3F1, had an apparent increase intelomere length4. It has recently been sug-gested that aging is a price paid by cells forp53-mediated tumor suppression13. It willbe interesting to learn whether theincreased incidence of tumors in clonesreflects the amelioration of aging con-comitant with a depression in levels of p53and other tumor suppressors, or whetherthe tumorigenic phenotype is sympto-matic of immune impairment.

In sum, this latest report6 suggests thatthe organismal sequelae of nuclear-transfer cloning can be subtle, long-termand specific. As such, it provides what isperhaps a paradoxical hope that thefidelity of the process may be improved.In establishing the areas that need to beaddressed to achieve this, the report her-alds a new horizon in the study ofnuclear-transfer cloning. �1. Wakayama, T., Perry, A.C.F., Zuccotti, M., Johnson,

K.R. & Yanagimachi, R. Nature 394, 369–374 (1998).2. Inoue, K. et al. Science 295, 297 (2002).3. Wakayama, T. & Yanagimachi, R. Nature Genet. 22,

127–128 (1999).4. Wakayama, T. et al. Nature 407, 318–319 (2000).5. Tamashiro, K.L.K., Wakayama, T., Blanchard, R.J.,

Blanchard, D.C. & Yanagimachi, R. Biol. Reprod. 63,328–334 (2000).

6. Ogonuki, N. et al. Nature Genet. 30, 253–254 (2002).7. Renard, J.P. et al. Lancet 353, 1489–1491 (1999).8. Wakayama, T. & Yanagimachi, R. Mol. Reprod. Dev.

58, 376–383 (2001).9. Wakayama, T., Rodriguez, I., Perry, A.C.F.,

Yanagimachi, R. & Mombaerts, P. Proc. Natl Acad.Sci. USA 96, 14984–14989 (1999).

10. Eggan, K. et al. Proc. Natl Acad. Sci. USA 98,6209–6214 (2001).

11. Willadsen, S.M. Nature 320, 63–65 (1986).12. Ogura, A., Inoue, K., Takano, K., Wakayama, T. &

Yanagimachi, R. Mol. Reprod. Dev. 57, 55–59 (2000).13. Tyner, S.D. et al. Nature 415, 45–53 (2002).

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