BIOLOGY OF REPRODUCTION 66, 1649–1655 (2002)
Remarkable Differences in Telomere Lengths among Cloned Cattle Derived
from Different Cell Types1
Norikazu Miyashita,2,4 Kazuho Shiga,5 Miharu Yonai,6 Kanako Kaneyama,6 Shuji Kobayashi,6
Toshiyuki Kojima,6 Yuji Goto,7 Masao Kishi,8 Hisashi Aso,3,4 Toshiyuki Suzuki,9 Minoru Sakaguchi,10
and Takashi Nagai11
Laboratory of Cellular Biology,4 National Institute of Animal Industry, Kukisaki, Ibaraki 305-0901, Japan
Department of Beef Cattle Production Technology,5 Oita Prefectural Livestock Experiment Station, Kujyu,
Oita 878-0201, Japan
Department of Technology,6 National Livestock Breeding Center, Nishigo, Fukushima 961-8511, Japan
Tokachi Station,7 National Livestock Breeding Center, Otofuke, Hokkaido 080-0572, Japan
Embryo Transplantation Laboratory,8 Snow Brand Milk Products Co., Ltd., Tomakomai, Hokkaido 059-1365, Japan
Animal Industry Research Institute,9 Iwate Prefectural Agriculture Research Center, Takizawa, Iwate 020-0173, Japan
Department of Animal Production,10 Hokkaido National Agricultural Experiment Station, Sapporo,
Hokkaido 062-8555, Japan
Embryonic Technology Laboratory,11 National Institute of Agrobiological Sciences, Kukisaki, Ibaraki 305-8602, Japan
length among donor cells and more or less elongation of telomere lengths induced by cloning.
ABSTRACT
Regarding cloned animals, interesting questions have been
raised as to whether cloning restores cellular senescence undergone by their donor cells and how long cloned animals will
be able to live. Focusing our attention on differences in telomere
lengths depending on the tissue, we had produced 14 cloned
cattle by using nuclei of donor cells derived from muscle, oviduct, mammary, and ear skin. Here, we show remarkable variation in telomere lengths among them using Southern blot analysis with telomere-specific probe. Telomere lengths in cloned
cattle derived from muscle cells of an old bull were longer than
those of a donor animal but were within the variation in normal
calves. On the other hand, those derived from oviductal and
mammary epithelial cells of an equally old cow were surprisingly shorter than any found in control cattle. The telomere
lengths of cloned cattle derived from fibroblasts and oviductal
epithelial cells of younger cattle showed the former and the latter results, respectively. In both cases, however, less telomere
erosion or telomere extension from nuclear transfer to birth in
most cloned cattle was observed in comparison with telomere
erosion from fertilization to birth in control cattle. Embryonic
cell-cloned cattle and their offspring calves were also shown to
have telomeres longer than those in age-matched controls. These
observations indicate that cloning does not necessarily restore
the telomere clock but, rather, that nuclear transfer itself may
commonly trigger an elongation of telomeres, probably more or
less according to donor cell type. Remarkable variations among
cloned cattle are suggested to be caused by variation in telomere
Funded by the 21st Century Green Frontier, Clone Project of the Ministry
of Agriculture, Forestry & Fisheries of Japan. This work was supported by
the Department of Animal Breeding and Reproduction in National Institute of Livestock and Grassland Sciences.
2
Correspondence and current address: Norikazu Miyashita, Animal Cell
Biology Laboratory, National Institute of Agrobiological Sciences, Kukisaki, Ibaraki 305-8602, Japan. FAX: 81 298 38 8689;
e-mail: nmiya@affrc.go.jp
3
Current address: Laboratory of Functional Morphology, Tohoku University, Aoba, Sendai 981-8555, Japan.
1
Received: 22 August 2001.
First decision: 25 September 2001.
Accepted: 20 December 2001.
Q 2002 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
aging, embryo, female reproductive tract, mammary glands,
sperm
INTRODUCTION
Somatic cell-cloned animals, which are produced by nuclear transfer of cultured somatic cells into enucleated oocytes, have so far been produced from sheep [1, 2], mice
[3–5], cattle [6–10], and pigs [11–13]. Regarding cloning
strategies in the future, analysis of basic biological mechanisms of cloning will predominate in the cloning of small
laboratory mammals, and practical uses and benefits will
determine the extent of cloning of large farm animals. In
particular, the duplication of high-performance animals and
contributions to efficient breeding schemes have been pursued in livestock production. Additionally, animal cloning
using cultured somatic cells fortunately offers the possibility of transgenic or gene-targeted livestock production for
animal factories without embryonic stem cells that are not
yet available except for mice [14–16].
It is important, however, that livestock animal clones be
used not only for practical applications but also for research
to elucidate the mechanisms of cloning. Because many
questions regarding cloned animals remain unsolved (e.g.,
male clones that can be produced with difficulty in mice
[4] can be produced easily in cattle [8]), comparative research among clones of several species are essential. For
breeding schemes in Japan, all bulls and many cows undergo a progeny test, and genetic potentials in bulls and
cows are estimated statistically using their progeny’s data
regarding many traits, such as growth, milk and meat production. Additionally, their lineage is also recorded retroactively to several generations. Because most cloned cattle
in Japan were produced from such informative cattle and
the identity test between donor cattle and their clones has
already started, we will gain favorable knowledge regarding
how to solve the questions of cloning in the future. Furthermore, by accumulation of comparative research among
clones of several species, mechanisms acting in the process
of this promising technique of cloning will gradually be
revealed.
Interesting questions have been raised as to how old
1649
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MIYASHITA ET AL.
pulse-field electrophoresis to separate long telomeric DNAs [28–30] using
a program designed to ensure good separation for size between 1 and 50
kb with the FIGE Mapper electrophoresis system (Bio-Rad, Hercules,
CA). The DNA was then transferred to nylon membranes by a standard
Southern blotting procedure. DNA was cross-linked to the membrane with
1200 mJ of ultraviolet light.
Hybridization
FIG. 1. Linear regression of telomere lengths showing the decline in
telomere lengths with age for 50 control cattle and positions of telomere
lengths in the cloned cattle and their donor cells. Dots indicate leukocytes
in control cattle, and a solid line indicates linear regression of telomere
lengths. Closed symbols indicate cultured donor cells, and open symbols
indicate leukocytes of their cloned calves. Squares indicate oviductal epithelial cells from an old Holstein and a Jersey cow and leukocytes of
their cloned calves. Diamonds indicate mammary epithelial cells from
the same Holstein cow and leukocytes of their cloned calves. A cross
indicates leukocytes, sampled at 14 yr of age, of an old Japanese Black
bull that provided muscle cells for cloning. Circles indicate muscle cells,
sampled at 12 yr of age, of the Japanese Black bull and leukocytes of its
cloned cattle. Triangles indicate skin fibroblasts from another Japanese
Black bull and its cloned calves.
The telomere-specific oligonucleotide (TTAGGG)3 was end-labeled at
378C for 15 min using terminal deoxynucleotidyl transferase from the DIG
Oligonucleotide 39-End Labeling Kit (Roche, Mannheim, Germany). The
blotted nylon membranes were prehybridized in 40 ml of DIG Easy Hyb
(Roche) for 2 h at 378C, and then were hybridized in 10 ml of DIG Easy
Hyb containing 50 pmol of end-labeled, telomere-specific probe for 16 h
at 378C. Membranes were washed 3 times in 50 ml of 0.53 standard saline
citrate (SSC; 13 SSC: 0.15 M NaCl plus 0.015 M sodium citrate) for 15
min at 378C. Chemiluminescence was performed using the DIG Luminescent Detection Kit (Roche) and exposed to x-ray film (RX-U; Fuji Photo
Film, Minamiashigara, Japan).
Calculation of Telomere Length
Telomere length was calculated as described by Tian et al. [24] and
Norwood and Dimitrov [29]. The chemiluminescence signal was acquired
by a Model GS-700 Imaging Densitometer (Bio-Rad) at the highest resolution of 0.01 mm. The acquired 16-bit images were then processed by
using the Molecular Analyst software (Bio-Rad). Once the distance calibration was obtained, all distances were converted to telomere lengths,
and the dependence of the signal intensity on the telomere length at each
point (0.01 mm) was analyzed. Mean terminal restriction fragment (TRF)
size was determined as
S(ODi · Li)/S(ODi)
cloned animals are in terms of genetic age; how long they
will be able to live and stand for milk production, reproduction, and so on; and in particular, when to use donor
cells derived from old animals. It has been reported in the
cloned sheep ‘‘Dolly’’ that the telomere length, which
shortens with each cell division, resulting in cellular senescence [17–21], is consistent with the age of the mammary
tissue used for nuclear transplantation from the 6-yr-old donor sheep and that the length is significantly shorter than
that in age-matched control sheep [22]. This suggests that
Dolly may not achieve a full, normal life span. On the other
hand, in cases of cloned cattle produced from near-senescent fibroblasts, extension of telomere lengths, in which
telomere lengths in cloned cattle were the same as or longer
than those in newborn normal calves, has been reported
[23]. Tian et al. [24], Wakayama et al. [5], and Betts et al.
[25] have also reported normal telomere lengths in clone
animals derived from fibroblasts and cumulus cells. Therefore, the question of the discrepancy between Dolly’s shortened telomere and others’ restored telomeres remains.
The present study was conducted to determine the effects
of cloning on telomeres. Focusing our attention on differences in telomere lengths depending on the tissue [26, 27],
we had produced cloned cattle from reconstructed embryos
with nuclei of epithelial cells from oviduct and mammary
gland, skin fibroblasts, and muscle cells [8, 9]. In the present study, we examine variation in telomere lengths in
these cloned cattle, and we discuss the effect of cloning on
telomere length.
MATERIALS AND METHODS
DNA Isolation and Southern Blot Analysis
The DNA from leukocytes or cultured cells was prepared with the
SDS-phenol-proteinase K extraction methods, and equal amounts of DNA
(1 mg) were digested by both the restriction enzyme AluI and HinfI. Samples, 1-kilobase (kb) ladder marker, and 5-kb ladder marker were then
loaded on a 1% (w/v) agarose gel (15 3 15 cm). The gels were run by
where ODi is signal intensity and Li is the telomere length at each point i
(0.01 mm) within the lane. Sums were calculated over the range 3–50 kb.
Culture of Senescent Cells
To determine a critical telomere length in bovine somatic cells, telomere erosion was examined in culture using a clonal bovine intramuscular
preadipocyte (BIP) line [31]. The BIP cells were cultured in Dulbecco
modified Eagle medium (Gibco BRL, Grand Island, NY) plus 10% fetal
bovine serum and antibiotics at 378C in a humidified atmosphere of 5%
CO2 and 95% air. For each passage, cells were cultured until confluency,
were disaggregated by incubation in a 0.02% trypsin and 0.01% EDTA
solution, and were allocated to new dishes for further passage. Normally,
each passage lasted approximately 3 days. The cell line was maintained
routinely until the cells exhibited features of replicative senescence and
the number of cells could no longer increase.
Statistical Analysis
The mean TRF analyses in cloned cattle were repeated at least 2 times,
and the average is shown in this paper. Because the methods for measuring
mean TRF sizes were reported to have an error of approximately 0.3 kb
in humans [30] and telomeres in cattle were demonstrated to be 2 times
longer than those of humans in this study, when differences between two
measurements for the same sample were beyond 0.5 kb, more measurements were carried out until the difference between two measurements
was within 0.5 kb. Linear regression of mean TRF sizes in 50 normal
Japanese Black cattle against age were analyzed with the least-squares
method. Differences among mean TRF sizes in the donor cells, cloned
cattle, control cattle, and control sperm were analyzed by Student t-test.
RESULTS
Telomere Lengths in Normal Cattle and Senescent Cells
To calibrate the telomere clock in normal cattle, telomere
erosion in vivo was analyzed using leukocytes from 50 normal Japanese Black cattle (age, 0–18 yr; Fig. 1). In newborn calves, the mean sizes of the terminal telomere fragments obtained by cutting with restriction enzymes (i.e.,
mean TRF) were found to be 19.0–21.9 kb (20.43 6 0.28
kb), whereas in two 18-yr-old animals, which were regard-
TELOMERE LENGTH VARIATIONS IN CLONED CATTLE
ed as aged, the mean TRF sizes were 15.1 and 16.8 kb
(Fig. 2A). Then, the analysis yielded a significant linear
regression of telomere lengths (LRTL): The mean TRF size
regression was 20.23 kb/yr 1 20.54 kb (r2 5 0.56, P ,
0.01). The mean TRF sizes of bovine spermatozoa were
22.1–23.9 kb (23.0 6 0.28 kb) (Fig. 2A). Because the mean
TRF sizes of spermatozoa in humans are kept longer than
those of any tissues beyond generation and then hypothesized as initial lengths of telomere erosion [19], telomere
erosion from the beginning of fertilization to birth in cattle
was also, thus, revealed to be significantly apparent (P ,
0.01) and approximately 2.6 kb. On the other hand, to determine a critical telomere length in bovine somatic cells,
telomere erosion in culture was examined using a clonal
BIP line that exhibited a fibroblastic appearance and abundant replicative capacity [31]. After 60 passages of culture,
BIP cells exhibited features of cellular senescence and almost arrested their proliferation. In such senescent cells, the
mean TRF size had also stopped decreasing at 9.7 kb (Fig.
2A).
Telomere Lengths in Cloned Cattle Derived
from Epithelial Cells of an Old Cow
We produced 5 and 1 cloned cattle by nuclear transfer
of cultured oviductal epithelial cells and mammary epithelial cells, respectively, from a 13-yr-old Holstein cow [9]
(Table 1). The mean TRF sizes in all 6 newborn cloned
calves were significantly smaller (P , 0.01) than those in
age-matched controls and surprisingly smaller than those in
18-yr-old control animals (Figs. 1 and 2B). In terms of the
age-dependent LRTL, estimated ages taken from leukocytes
of cloned calves derived from Holstein oviductal and mammary epithelial cells (as an index of aging not of the whole
body but, rather, only of leukocytes) were surprisingly old
and approximately 30–35 and 29 yr, respectively (Fig. 3),
regardless of the actual bovine lifetime of 15–20 yr at most.
However, all of them remained alive and normal at 22–25
mo of age, except that one derived from mammary epithelial cells was slaughtered for deformity. On the other hand,
the mean TRF sizes of their donor cells were also revealed
to be much shorter than those of leukocytes in age-matched
controls in LRTL terms. Telomere erosions from nuclear
FIG. 2. Representative analysis of telomeres in cloned cattle. Telomeres
of cells for which sources are not described are from leukocytes. A) Telomeres in control spermatozoa (lane 1), calf (lane 2), 18-yr-old cattle (lane
3), and senescent cultured cells (lane 4) were demonstrated. B) Telomeres
in cloned cattle (lanes 6, 7, 9, and 10) produced from cultured oviductal
epithelial cells (lanes 5 and 8) were demonstrated. Lanes 5–7 were derived from a 6-yr-old Jersey cow. Lanes 8–10 were derived from a 13-yrold Holstein cow. C and D) Telomeres in cloned calves (lanes 13, 14, 16,
and 17) produced from cultured muscle (lane 12) and skin fibroblast (lane
15) cells, respectively, were demonstrated. Lanes 12–14 were derived
from a 12-yr-old bull (lane 11). Lanes 15–17 were derived from a 2-yrold bull. E) Telomeres in embryonic cell-cloned cattle (lanes 19 and 20)
and her offspring calf (lane 21) were demonstrated. The telomere lengths
were much longer than those of a control calf (lane 18).
transfer to birth were revealed to be 1.5–2.7 kb (Table 1)
and were the same as or less than telomere erosion from
fertilization to birth in normal cattle.
Telomere Length in Cloned Cattle Derived from Muscle
Cells of an Old Bull
The mean TRF sizes of leukocytes from 2 newborn
cloned calves that were produced by nuclear transfer of
cultured cells derived from the muscle tissue of a 12-yr-old
(as of August 1997) Japanese Black bull [8] were within
variation among the age-matched control calves (Figs. 1
and 2C). These cloned cattle remained alive and normal at
24–29 mo of age and had calves by artificial insemination
using their spermatozoa. The mean TRF sizes of their donor
cells, regardless of 6 passages of culture before nuclear
transfer, were be much longer than those of leukocytes from
TABLE 1. Telomere lengths of somatic cell-cloned cattle and telomere length change from nuclear transfer to birth.
Donor cell
type and mean
TRF (kb)
Cloned cattle
ID
1651
Mean TRF (kb)
15.2 (Oviductal epithelial cells from a 13-yr-old Holstein cow)
ovh1
12.9
ovh2
13.4
ovh3
13.2
ovh4
13.7
ovh5
12.5
16.3 (Mammary epithelial cells from a 13-yr-old Holstein cow)
meh1
14.3
16.9 (Oviductal epithelial cells from a 6-yr-old Jersey cow)
ovj1
15.9
ovj2
14.9
ovj3
16.0
ovj4
14.9
20.1 (Muscle cells from a 12-yr-old Japanese Black bull)
mus1
19.6
mus2
19.9
18.2 (Skin fibroblasts from a 2-yr-old Japanese Black bull)
ear1
20.0
ear2
20.4
Telomere length
change (kb)
Current status
22.3
21.8
22.0
21.5
22.7
Live
Live
Live
Live
Live
22.0
Slaughter for deformity
21.0
22.0
20.9
22.0
Live
Live
Live
Live
20.5
20.2
Live
Live
11.8
12.2
Slaughter for research
Died of dystocia
1652
MIYASHITA ET AL.
those in age-matched controls and surprisingly smaller than
those in 18-yr-old control animals (Figs. 1 and 2B). Telomere erosions from nuclear transfer to birth were 0.9–2.0
kb (Table 1) and were less than telomere erosion from fertilization to birth in normal cattle. On the other hand, the
mean TRF sizes of leukocytes from 2 cloned cattle that
were produced by nuclear transfer of cultured skin cells
derived from an ear of a 2-yr-old Japanese Black bull and
their donor cells were compared with those of leukocytes
obtained from age-matched controls. The mean TRF sizes
in the newborn cloned calves were within variation among
the age-matched control calves, just as in cloned cattle derived from muscle cells of an old bull (Figs. 1 and 2D).
One of the cloned calves was very heavy in body weight
(63 kg) compared to the standard body weight for male
calves of this breed (20–30 kg) and died shortly after birth
because of dystocia at parturition; the other cloned calf was
normal in body weight (27 kg) and slaughtered for histopathological research (Table 1). Regardless of a few passages of culture before nuclear transfer, the mean TRF sizes
of donor cells from ear skin were to be much smaller than
those of leukocytes in the newborn cloned calves. Thus,
telomere extension might have occurred from nuclear transfer to birth. The extent of changes in telomere lengths from
nuclear transfer to birth was significantly different among
their donor cell types of epithelial cells, muscle cells, and
fibroblasts (P , 0.01).
Telomere Length in Embryonic Cell-Cloned Cattle
FIG. 3. Cloned cattle produced by nuclear transfer of cultured oviductal
epithelial cells from a 13-yr-old Holstein cow and a 6-yr-old Jersey cow.
A) Holstein clones at 12 mo of age. B) Jersey clones at 10 mo of age.
cattle at the corresponding age in LRTL terms. Therefore,
less telomere erosion from nuclear transfer to birth was
revealed in comparison with that from fertilization to birth
in normal cattle.
Telomere Length in Cloned Cattle Derived from Cultured
Cells of Younger Cattle
We produced 4 cloned cattle by nuclear transfer of cultured oviductal epithelial cells from a 6-yr-old Jersey cow
[9]. Like cloned calves derived from epithelial cells of an
old Holstein cow, the mean TRF sizes in all 4 newborn
cloned calves were significantly smaller (P , 0.01) than
We investigated telomere lengths in embryonic cellcloned cattle that had been produced by nuclear transfer of
the nuclei of 28- to 49-cell stage blastomeres to enucleated
oocytes. All of 6 embryonic cell-cloned cattle we tested
had significantly longer telomeres than age-matched controls (P , 0.01), and their offspring calves, which were
obtained by artificial insemination of normal spermatozoa
to female embryonic cell-cloned cattle, also had telomere
lengths somewhere in between those of age-matched control calves and embryonic cell-cloned cattle (Fig. 2E and
Table 2).
DISCUSSION
To our knowledge, the present study is the first to show
that remarkable variation exists among telomere lengths in
cloned cattle produced from donor cells derived from 4
different kinds of tissue. Particularly, telomere lengths in
TABLE 2. Telomere lengths and cloning data of embryonic cell-cloned cattle and their offspring.
Clone ID
Sex
Embryonic cell-cloned cattle
E61
M
E62
M
E63
M
EC1
F
EC2
F
EC3
F
Offspringb
ES1c
M
ES2d
M
a
Breed
Days postcoitum
of donor cell (day)
Development stage
of donor cell (cell)
Mean TRF
(kb)
JBa
JB
JB
JB
JB
JB
5.0
5.0
5.0
5.5
5.5
5.0
34
34
34
49
49
28
23.8
23.3
22.7
21.7
22.6
26.2
JB
JB
24.6
21.0
JB, Breed of Japanese Black.
Offspring were obtained by artificial insemination of normal spermatozoa to female embryonic cell-cloned cattle.
c Dam of ES1 was EC3.
d Dam of ES2 was EC2.
b
TELOMERE LENGTH VARIATIONS IN CLONED CATTLE
cloned calves derived from epithelial cells were surprisingly shorter than those in all control cattle that we tested. In
the case of Dolly, the mean TRF size was reported to be
consistent with those of her donor cells (i.e., cultured mammary epithelial cells) and smaller than those of the agematched control sheep [22]. Lanza et al. [23] reported that
the mean TRF sizes in cloned cattle produced from senescent fibroblasts were much longer than those in their donor
cells, and those authors concluded that cloning using bovine fibroblasts restores the telomere clock. It was suggested that the difference between Dolly and their cloned
cattle might be due to species differences and/or to differences in nuclear transfer techniques or donor cell types
[23]. Furthermore, Tian et al. [24] reported that telomere
lengths in cloned cattle derived from fibroblasts and cumulus cells were normal and pointed out that Dolly’s short
telomeres likely are an exception, but without referring to
the different cell types used as donor cells. However, our
results showed both remarkably short telomere lengths in
cloned cattle derived from epithelial cells and normal telomere lengths in cloned cattle derived from muscle cells and
fibroblasts. Therefore, it can be suggested that Dolly’s short
telomeres are not an exception but, rather, that different
donor cell types might lead to different telomere lengths in
the resultant cloned cattle.
The number of samples in the present study was limited
and the conditions for comparison were inequitable, but the
most likely explanation for the remarkable variation among
telomere lengths in our cloned cattle was that it reflected
the telomere length variation of their donor cells and the
extent of telomere length change induced by cloning. First,
the variation among donor cells may be caused by cell typespecific factors in vivo, e.g., frequencies of cell division in
each tissue and intracellular environments such as a concentration of superoxide that is detrimental to DNA helices,
and so on. It was suggested that the telomere erosion rate
of the oviductal and mammary epithelial cells in vivo may
be much more rapid than that in leukocytes, and that the
extremely shortened telomere lengths of resultant donor
cells may lead to the remarkably short telomere lengths of
the cloned calves. In contrast, it was suggested that telomere lengths of the muscle cells could be retained even in
the old donor bull, because the donor cells were possibly
derived from muscle satellite cells that play a role in restoring injured muscle tissue and are inactive for cell division except when performing that function. Therefore,
telomere lengths of the muscle cells might lead to a longer
mean TRF of leukocytes in the cloned calves than that in
the donor bull. Second, it was also suggested that the effect
of aging in vivo of donor cattle might contribute to the
telomere length variation of donor cells, because the difference between telomere lengths in oviductal epithelial
cells from 2 cows in the present study was reflected by the
different ages of the cows. Furthermore, the difference
might result in telomere length variation among cloned cattle that is significantly regressive (r2 5 0.840, P , 0.01).
The possibility that telomere length variation of donor cells
was contributed to by the genetic background of the donor
cattle was not excluded, because variation among telomere
lengths in the leukocytes of control cattle was not so small.
On the other hand, telomere length variation among donor
cells might not be induced by culture so much, because the
donor cells were used after only a few passages.
Not only initial telomere lengths in donor cells but also
the extent of telomere length change induced after cloning
might lead to a remarkable variation among telomere
1653
lengths in cloned cattle. In all previous reports except the
case of Dolly [22], it was demonstrated that shortened telomeres in donor fibroblasts and cumulus cells both extended
and restored from nuclear transfer to birth; therefore, normal telomere lengths in cloned calves were obtained [23–
25]. Betts et al. [25] pointed out that the rebuilding of shorter telomere lengths in donor cells could be due to telomerase activities, which are detected as early as the blastocyst
stage in reconstructed embryos. The present study showed
that some cloned cattle had longer telomere lengths than
those of their donor cells, and that others had telomere
lengths just a bit shorter than those of their donor cells.
Additionally, the extent of telomere length changes from
nuclear transfer to birth were significantly different among
donor cell types (P , 0.01). It is of interest that less telomere erosion or telomere extension from nuclear transfer
to birth was observed in most cloned cattle in comparison
with telomere erosion from fertilization to birth in control
cattle (2.6 kb). Thus, it was suggested that cloning does not
necessarily restore the telomere clock but, rather, may commonly trigger an elongation of telomere lengths, probably
more or less according to cell types. Not only in cattle but
also in humans, telomere lengths were reported to decrease
from 22 kb in spermatozoa to 10 kb in newborn-baby leukocytes [17]. Hence, it is most probable that telomeres also
decrease from fertilization to birth in sheep. If so, then Dolly’s telomere length, which was the same as that of the
donor mammary epithelial cell, might elongate transiently
after cloning, resulting in compensation for the loss during
embryo-fetus development.
The exact explanation for the difference among donor
cell types in the extent of telomere length elongation from
nuclear transfer to birth is unclear at this moment. In humans, it was reported that telomerase activities are not detected in most somatic cells except for a part of the stem
cells and fetal cells [32–34], and that telomerase activities
even in telomerase-positive somatic cells are lost quickly
by culture and particularly during passage [35]. In cattle,
similar results were obtained [25], and telomerase activities
in oviduct, mammary, muscle, and ear skin tissue were very
weak or not detected in our study (data not shown). Thus,
it was suggested that the difference among donor cell types
in telomere length elongation was not caused by telomerase
activities taken from donor cells. It may, instead, be partially explained by a possibility that donor cell type-specific
sensitivities to telomerase, based on telomere-binding proteins [36, 37], lead to the difference in the extent of telomere elongation in reconstructed embryos. For example,
telomere-binding protein TRF1, which inhibits telomerase
activities, was expressed differentially according to the tissue type [37].
To confirm whether telomere elongation is only specific
to somatic cell-cloned cattle, we investigated telomere
lengths in embryonic cell-cloned cattle that had been produced by nuclear transfer of blastomeres to enucleated oocytes. They are regarded as being homogeneous to normal
cattle, because totipotent cells in germline were used as
their donor cells. All of 6 embryonic cell-cloned cattle and
their offspring calves that we tested had significantly (P ,
0.01) longer telomeres than the age-matched controls.
These results might indicate that elongation of telomeres
did not result from cloning with ‘‘somatic’’ cells but, rather,
was induced by the technique of nuclear transfer itself.
To determine a critical telomere length in bovine somatic
cells, telomere erosion was examined in culture using a
clonal BIP cell line, in which the mean TRF size had also
1654
MIYASHITA ET AL.
stopped decreasing at 9.7 kb. This critical length is longer
than that from human fibroblast [20] and leukocytes [38]
but is shorter than the telomere lengths of sterile mice derived from successive crossing of mice lacking telomerase
RNA and exhibiting decreased proliferation in highly proliferative organs [39, 40]. Because the mean TRF sizes of
leukocytes in old cattle were much longer than the critical
length found in the BIP line, it may be difficult to relate
telomere erosion to senescence in normal cattle. Therefore,
it was indicated that cloned calves derived from epithelial
cells could live regardless of the ages estimated from telomeres in leukocytes, which were surprisingly old at approximately 24–33 yr. However, it is possible that cloned
cattle produced from oviductal and mammary epithelial
cells may lose function sooner in tissues with cells regulated at a high rate of telomere erosion and, consequently,
die young because of their remarkably short telomeres.
It is often said that we must take care of the decreased
genetic variation in the livestock population and the resultant increased risk of inbreeding depression and genetic disease induced by the overdiffusion of cloning. Likewise, it
may be important to be careful of telomere lengths, not only
for individual cloned animals but also for whole species. If
cloned animals and their offspring that had short telomeres
were selectively reproduced for their high performance and
disseminated without due care, the whole species might become short-lived and, in some cases, face extinction. When
applying cloning techniques in the livestock industry, we
must check telomere lengths and take them into account,
for the sake of longevity, when making mating plans.
ACKNOWLEDGMENTS
We thank Y. Nagamine for statistical analysis and helpful advice, Y.
Sasae and Y. Yamada for insightful discussions and providing samples,
and T. Takenouchi, M. Higuchi, and Y. Shingai for advice and assistance.
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