Success of cloning using adult somaticcells has been reported in sheep1,mice2,3 and cattle47. The report thatDolly8 the sheep, the first clone from anadult mammal, inherited shortenedtelomeres from her cell donor and that hertelomeres were further shortened by thebrief culture of donor cells has raised seri-ous scientific and public concerns aboutthe genetic age and potential develop-mental problems of cloned animals. Thisobservation was challenged by a recentreport9 that showed calves cloned fromfetal cells have longer telomeres than theirage-matched controls. The questionremains whether Dollys short telomereswere an exception or a general fact, whichwould differ from the telomeres of fetal-derived clones.

We have cloned 10 calves from a 13-year-old dairy cow using fibroblast andcumulus cells (manuscript in prepara-tion). Of the clones, 6 died shortly afterbirth and the remaining 4 are now 1213months of age, appearing healthy andindistinguishable from their naturallyreproduced peers. To examine the telom-ere lengths of our clones, we performedterminal restriction fragment (TRF)

analyses10 using genomic DNA from eartissue samples collected from all 10 clones,4 age-matched controls and the donorcow. We used ear tissue because cells takenfrom the ear were used for nuclear trans-fer, and cell division in the ear is affectedonly by the growth of the animals.

We found that telomere lengths of thefour live clones were not different fromthose of their age-matched controls(15.380.62 versus 14.730.49 kb, P>0.05;Fig. 1a,b), and all calves had longer telom-eres than the donor cow (12.430.49 kb,P0.05; Fig. 1a,b).Our data demonstrate that telomeres areshorter in the aged cow, and that the short-ened telomeres of the donor somatic cellsare restored to normal length after nucleartransfer, regardless of the viability of clonesafter birth. This suggests that the frequentneonatal death of cloned animals is notlikely to be caused by short telomeres.

We further tested possible telomereshortening in donor fibroblast cells duringin vitro culture (Fig. 1c). Regression analy-sis indicates that gradual reduction in

telomere lengths occurred in culture(r=0.91, P

Camurati-Engelmann disease (CED;MIM 131300), or progressive dia-physeal dysplasia, is a rare, sclerosing bonedysplasia inherited in an autosomal domi-nant manner. Recently, the gene causingCED has been assigned to the chromoso-mal region 19q13 (refs 13). Because thisregion contains the gene encoding trans-forming growth factor-1 (TGFB1), animportant mediator of bone remodelling4,we evaluated TGFB1 as a candidate genefor causing CED.

CED is manifested by severe pains in thelegs, muscular weakness and a waddlinggait. Further clinical symptoms include easyfatiguability, reduced muscle mass, generalweakness, exophthalmos, facial paralysis,hearing difficulties and loss of vision. Radi-ologically, the disease is characterized by afusiform thickening of the diaphyseal andoccasionally the metaphyseal cortex of thelong bones, narrowing of the medullarycanal and sclerosis at the skull base.

Here we screen TGFB1 for mutations

using patient material from six CED fami-lies. Besides the previously reportedextended Israeli family2, we included fivesmaller families of European origin. Muta-tion analysis revealed four different muta-tions in these six families (Fig. 1). Theseinclude 3 missense mutations resulting in aR218C substitution in families 13, aC225R substitution in family 4 and a Y81Hsubstitution in family 5. Finally, a repeat of6 leucines located in the signal peptide atthe amino-terminal end of the TGF-1peptide is extended to 9 leucines in family6. None of the mutations were found in200 control chromosomes.

TGF-1 is synthesized as a large precur-sor molecule that is processed in the Golgiapparatus, including glycosylation, cleav-age of the signal peptide and dimerizationby disulphide bonds (Fig. 1b). Next, the

the previous study versus 10 clones usedhere; and (iii) different sources of DNAwere used in the TRF assays. White bloodcells, used in the previous study, can bestimulated to divide by infection. The TRFlengths we report here are in agreementwith those reported for cattle (13.5 kb forkidney cells and 15.3 kb for sperm; ref. 15).Our study also differs from that of Lanza etal.9. They examined telomere lengths infetal-derived clones, whereas we investi-gated those in adult-derived clones. Theyfound that the fetal clones had longertelomeres than aged-matched controls,whereas we found normal telomere lengthsin our adult-derived clones. The differencesare probably due to different sources of thedonor cells and different culture conditions.The donor cells in our study were culturedonly briefly, whereas those in the study byLanza et al.9 were cultured to near senes-

cence. Over-compensation in telomerereprogramming is a possibility when near-senescent cells were used for cloningbecause they are near the limit of telomereshortening. Very recently, telomere restora-tion has also been observed in cloned mice,with no sign of incremental erosion afterserial cloning up to six generations16.Therefore, the shortened telomeres foundin Dolly are likely to be an exception,whereas the restored telomere patternfound in cattle and mice may be the generalresult for cloned animals.

It is unlikely that the cloning procedureselected a subset of the donor nuclei thathad much longer telomeres than the meanof donor nuclei, thus resulting in theapparent restoration where there was none.Telomere elongation has been observed9

after cloning of near-senescent cells. Addi-tionally, we have compared clones derived

from different age donors (from fetus to 17years of age) and found similar telomerelengths in all cloned calves irrespective ofthe donor age (manuscript in preparation).We thus conclude that adult-derived cloneshave normal telomere lengths, and thatrestoration of telomere lengths aftercloning likely occurs during embryogenesisthrough increased telomerase activity.

AcknowledgementsWe thank M. Barber and M. Taneja for cellculture and tissue biopsies, and R. Foote forcritical reading of the manuscript. This researchwas supported in part by a grant from theConnecticut Innovations, Inc.

X. Cindy Tian, Jie Xu & Xiangzhong Yang

Department of Animal Science and the

Biotechnology Center, University of Connecti-

cut, Storrs, Connecticut, USA. Correspondence

should be addressed to X.Y. (e-mail:

Xiangzhong.Yang@uconn.edu).

1. Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. &Campbell, K.H.S. Nature 385, 810813 (1997).

2. Wakayama, T., Perry, A.C.F., Zuccotti, M., Johnson,K.R. & Yanagimachi, R. Nature 394, 369374 (1998).

3. Rideout, W.M. III et al. Nature Genet. 24, 109110(2000).

4. Kato, Y. et al. Science 282, 20952098 (1998).5. Renard, J.-P. et al. Lancet 353, 14891491 (1998).6. Wells, D.N., Misica, P.M. & Tervit, H.R. Biol. Reprod.

60, 9961005 (1999).7. Kubota, C. et al. Proc. Natl Acad. Sci. USA 97,

990995 (2000).8. Shiels, P. et al. Nature 399, 316317 (1999).9. Lanza, R.P. et al. Science 288, 665669 (2000).10. Harley, C.B., Futcher, A.B. & Greider, C.W. Nature

345, 458460 (1990).11. Zhu, L. et al. Proc. Natl Acad. Sci. USA 95,

86488653 (1998).12. Bodnar, A.G. et al. Science 279, 349352 (1998).13. Betts, D.H. & King, W.A. Dev. Genet. 25, 397403

(1999).14. Xu, J. & Yang, X. Biol. Reprod. 63, 11241128

(2000).15. Kozik, A., Bradbury, E.M. & Zalensky, A. Mol.

Reprod. Dev. 51, 98104 (1998).16. Wakayama, T. et al. Nature 407, 318319 (2000).

brief communications

nature genetics volume 26 november 2000 273

Fig. 2 Relative telomerase activ-ity in embryos produced bynuclear transfer (NT) versus invitro fertilization (IVF). MOR,morula; BL, blastocyst. TRAP wasused to measure telomeraseactivity in lysates of earlyembryos at various stagesderived from cloning or in vitrofertilization using the TrapezeTelomerase Detection kit (Inter-gen) and analysed by QuantityOne 4.01 (Biorad). The experi-ment was repeated three timesand subjected to ANOVA andregression analyses.

Mutations in the gene encoding thelatency-associated peptide of TGF-1cause Camurati-Engelmann disease

2000 Nature America Inc. http://genetics.nature.com

2000

Nat

ure

Amer

ica

Inc.

ht

tp://

gen

etic

s.na

ture

.co

m