CLONING AND STEM CELLS

Volume 9, Number 1, 2007

Mary Ann Liebert, Inc.
DOI: 10.1089/clo.2006.0034

Endangered Wolves Cloned from Adult Somatic Cells

MIN KYU KIM,1 GOO JANG,1 HYUN JU OH,1 FIBRIANTO YUDA,1 HYE JIN KIM,1
WOO SUK HWANG,* MOHAMMAD SHAMIM HOSSEIN,* JOUNG JOO KIM,*
NAM SHIK SHIN,2 SUNG KEUN KANG,1 and BYEONG CHUN LEE1

ABSTRACT
Over the world, canine species, including the gray wolf, have been gradually endangered or
extinct. Many efforts have been made to recover and conserve these canids. The aim of this
study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for
conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and
cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes,
in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos
were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each
surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but
only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate
female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf.
In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of
wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach
for conserving endangered canids.
INTRODUCTION

HE REPORT THAT AN ADULT SHEEP had been

cloned (Wilmut et al., 1997) from the nucleus
of a frozen somatic cell fostered speculation that
cloning technologies might be applied to increase
the population size of endangered species, or
even to restore them after extinction. However,

unlike cloning of rodents and domestic animals,

because of the scarcity of oocytes, cloning of endangered or extinct species will require the use
of an alternative method such as intrageneric or
interspecies somatic cell nuclear transfer (SCNT).
Intrageneric SCNT has recently been applied to
some endangered species. A gaur (Bos gaurus) somatic cell was fused with an enucleated domes-

1Department

of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University,

Seoul, Korea.
2Zoo and Wild Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul, Korea.
*Current address: Sooam Biotech Research Foundation, Seoul, Korea.

130

ENDANGERED WOLVES CLONED FROM ADULT SOMATIC CELLS

tic cow (Bos taurus) oocyte and a live offspring

was born, although the calf died 2 days later (Vogel, 2001). An endangered argali sheep embryo
developed after nuclear transfer with domestic
sheep oocytes, and using the same approach, a
mouflon lamb was born (Loi et al., 2001).
Gray wolves (Canis lupus) are considered as a
threatened or endangered species in many countries including the Republic of Korea. In Korea,
gray wolves are rarely found in the wild, and a
small number of gray wolves have been brought
up in a special animal zoo. Recently, successful
birth of a cloned dog (Lee et al., 2005) by SCNT
raised the possibility of applying SCNT to conserve endangered or extinct canids including the
wolf and dingo. Here, we report the live birth of
two cloned gray wolves derived from nuclear
transfer of adult ear skin fibroblasts into enucleated dog oocytes.

MATERIAL AND METHODS

Care and use of animals
In this study, mixed breed female dogs from 1
to 3 years of age were used as oocyte donors and
embryo transfer recipients. The dogs were cared
for in facilities, and using procedures that exceeded the standards established by the Seoul
National University for Accreditation of Laboratory Animal Care. The study was conducted in
accordance with the Guide for the Care and Use
of Laboratory Animals in Seoul National University.

Chemicals
Unless otherwise indicated, all chemicals were
purchased from Sigma-Aldrich Corp. (St. Louis,
MO).

Vaginal cytology and progesterone levels

Female dogs were examined on a daily basis
for vulval swelling and serosanguinous discharge
from the starting day of natural heat. Smears were
obtained daily from the day of initial sign of proestrus to the day of surgery for oocyte retrieval.
Smears were collected by inserting a swab into
the lips of the vulva, then rolling it on a glass slide
and staining with a Diff-Quik (Sysmex, Kobe,
Japan) staining. In order to measure serum progesterone concentration, blood (35 mL) was col-

131

lected every day and centrifuged. Sera were analyzed using a DSL-3900 ACTIVE Progesterone
Coated-Tube Radioimmunoassay Kit (Diagnostic
Systems Laboratories, Inc., Webster, TX). The day
on which the progesterone concentration initially
reached 4.0 to 7.5 ng/mL was regarded as the day
of ovulation as described by Hase et al. (2000).

Donor cell preparation for SCNT

Adult fibroblasts were isolated from an ear
skin biopsy of a female gray wolf. Small pieces of
ear tissue fragment were washed three times in
D-phosphate buffered saline (PBS) and minced
with a surgical blade. The minced tissue was dissociated in Dulbeccos Modified Eagles medium
(DMEM, Invitrogen, Carlsbad, CA) supplemented with 0.25% (w/v) trypsin and 1 mM
EDTA (Invitrogen) for 1 h at 37C. Trypsinized
cells were washed once in Ca2!- and Mg2!-free
DPBS by centrifugation at 300g for 2 min, and
seeded onto 100-mm plastic culture dishes (Becton Dickinson, Lincoln Park, NJ). Seeded cells
were subsequently cultured for 6 to 8 days in
DMEM supplemented with 10% (v/v) fetal
bovine serum (FBS) (Invitrogen), 1 mM glutamine
(Invitrogen), 25 mM NaHCO3, and 1% (v/v) minimal essential medium nonessential amino acid
solution (Invitrogen) at 39C in a humidified
atmosphere of 5% CO2 and 95% air. After removal of unattached clumps of cells or explants,
attached cells were further cultured to confluency. These cells were subcultured at intervals of
4 to 6 days by trypsinization for 1 min using 0.1%
trypsin/0.02% EDTA, allocated to three new
dishes for further passaging and then stored in
freezing medium in liquid nitrogen at "196C using freezing medium. The freezing medium consisted of 80% (v/v) DMEM, 10% (v/v) dimethyl
sulfoxide, and 10% (v/v) FBS. Cells at passages 2
to 5 were used for SCNT. Prior to SCNT, cells
were thawed, cultured for 3 to 4 days until 100%
confluent, and retrieved from the monolayer by
trypsinization for 1 min.

Flushing of in vivo matured canine oocytes

Oocytes were retrieved from anesthetized female dogs by laparotomy. The fimbriated end of
the oviduct was accessed through the bursal slit
and cannulated using an inverted flanged bulb
needle. The needle was held in place by a surgical ligature, which was tied using a quick-release
device using a 3-cm plastic tube and hemostatic

132

forceps. The base of the oviduct, just above the

uterotubal junction, was visualized using digital
pressure to blanch the surrounding tissue and the
uterine tube lumen, and cannulated using a fine
hypodermic needle (24 gauge) attached to a syringe filled with embryo collection medium, consisting of HEPES-buffered tissue culture medium
(TCM)-199 supplemented 10% (v/v) FBS, 2 mM
NaHCO3, 5 mg/mL bovine serum albumin (BSA)
(Invitrogen). In vivo matured oocytes that were
obtained from flushing were transported to the
laboratory within 5 min in HEPES-buffered TCM199 at 38.5C.

Preparation of recipient oocytes for SCNT

Cumulus cells were removed from in vivo matured oocytes by repeated pipetting in 0.1% (v/v)
hyaluronidase (from bovine testis) in HEPESbuffered Ca2!-free CR2 medium (Rosenkrans et
al., 1993) with amino acids (hCR2aa). Oocytes
were then stained with 5 !g/mL bisbenzimide
(Hoechst 33342) for 5 min and observed under an
inverted microscope equipped with epifluorescence at 200# magnification. Each oocyte was
held with a holding micropipette (150 !m inner
diameter), then enucleated with a micromanipulator (Nikon-Narishige, Tokyo, Japan) in hCR2aa
supplemented with 10% (v/v) FBS and 5 !g/mL
cytochalasin B. The first polar body and adjacent
cytoplasm, containing the metaphase-II chromosomes, were removed using an aspiration pipette.
The enucleated oocytes were placed in TCM-199
supplemented with 10% (v/v) FBS and used for
SCNT.

Microinjection, fusion, activation,

and embryo culture
A single ear fibroblast from a wolf was deposited into the perivitelline space of an enucleated oocyte treated with 100 !g/mL phytohemagglutinin in hCR2aa to improve the
incorporation of the donor somatic cell with recipient cytoplast. Couplets were subsequently
placed in a fusion medium comprising 0.26 M
mannitol, 0.1 mM MgSO4, 0.5 mM HEPES, and
0.05% (w/v) BSA, and fused by using a needletype electrode. The single celloocyte couplet was
sandwiched between two parallel wires and attached to micromanipulators (Nikon-Narishige).
The contact surface between the cytoplast and the
donor cell was parallel to needle-type electrodes,
and electrical stimulation was delivered with an

KIM ET AL.

Electro-Cell Fusion apparatus (NEPA GENE Co.,

Chiba, Japan). The distance between the electrodes was approximately 180 !m (the diameter
of the oocytes). Two pulses of 72 V for a duration
of 15 !sec were applied, and the fusion of the
donor cell and the ooplast was observed under a
stereomicroscope 1 h after electric stimulation.
Only fused embryos were selected and cultured
for 3 h in modified synthetic oviductal fluid
(mSOF) as previously described (Jang et al., 2003).
The osmolarity and pH of mSOF were 270 to 280
mOsm and 7.2 to 7.3, respectively. Chemical activation of reconstructed canine embryos was induced by incubating embryos in mSOF containing 10 !M calcium ionophore for 4 min at 39C.
Reconstructed embryos were then washed and
further incubated for 4 h in mSOF supplemented
with 1.9 mM 6-dimethylaminopurine. After reconstruction, a group of five to six embryos was
cultured in 25-!L microdrops of mSOF overlaid
with mineral oil before embryo transfer.

Embryo transfer and pregnancy diagnosis

Within 4 h after reconstruction, cloned embryos were surgically transferred into the
oviducts of the surrogate mothers. Recipients
synchronized in natural estrus were used. For
surgical transfer, anesthesia was induced with 0.1
mg/kg acepromazine and 6 mg/kg propofol, and
general anesthesia was maintained with 2%
isoflurane. While in dorsal recumbency, recipients were aseptically prepared for surgery and a
caudal ventral incision was made to expose the
reproductive tract. Reconstructed embryos were
placed in the ampulla using a 3.5F Tom Cat Catheter (Sherwood, St. Louis, MO). Pregnancies were
detected using a SONOACE 9900 (Medison Co.
Ltd., Seoul, Korea) ultrasound scanner with an attached 7.0 MHz linear probe 23 days posttransfer. Pregnancy was monitored by ultrasound
every 2 weeks after initial confirmation.

DNA extraction and microsatellite

analysis for genotyping
Parentage analysis was performed in the nuclear donor fibroblasts, cloned wolves and surrogate recipients to confirm the genetic identity of
the offspring. Tissue fragments were obtained
from the tails of the two cloned wolves, and blood
samples were collected from the somatic cell
donor and the surrogate mothers. The tissue fragments, bloods, and trypsinized donor cells were

ENDANGERED WOLVES CLONED FROM ADULT SOMATIC CELLS

incubated with a lysis buffer [0.05 M Tris (pH 8.0),

0.05 M EDTA (pH 8.0), 0.5% sodium dodecyl sulfate] supplemented with 400 !g proteinase K
overnight, followed by phenol extraction and ethanol precipitation. Of the 27 microsatellites evaluated, the following 19 were selected for analysis: PEZ1, PEZ2, PEZ5, PEZ6, PEZ10, PEZ11,
PEZ12, PEZ13, PEZ15, PEZ17, REN105L03,
REN165M10, FH2140, FH2010, FH2054, CPH04,
CPH07, CHP14, and CPH22. The isolated genomic DNA samples were dissolved in 50 !L TE
and used for microsatellite assay with 19 specific
markers originally derived from dogs (Dolf et al.,
2000; Andersone et al., 2002; Vila et al., 2003; Lee
et al., 2005). Length variations were assayed by
polymerase chain reaction (PCR) amplification
with fluorescently labeled locus-specific primers
and polyacrylamide gel electrophoresis on an
automated DNA sequencer (ABI 373: Applied
Biosystems, Foster City, CA). Proprietary software (GeneScan and Genotyper; Applied Biosystems) was used to estimate PCR product size in
nucleotides.

Mitochondrial DNA analysis

Based on the complete nucleotide sequence of
canine mitochondrial DNA (mtDNA, GenBank
accession no. U96639), oligonucleotide primers
were synthesized for the hypervariable region
(L15,622-L16,030): forward, 5$-CATAGGACATATTAACTCAATC-3$; reverse, 5$-AAGTCCAGCTACAAGTTATTTG-3$. PCR amplifications were
conducted in a 50-!L volume containing 5 !L of
10# reaction buffer, containing 1.5 mM MgCl2, 0.2
mM dNTPs, 0.2 !M of primer, 1.5 U of Taq DNA

133

polymerase (Solgent, Daejeon, Korea), and 3050

ng genomic DNA. PCR was carried out for 3 min
at 95C, 35 cycles of 30 sec at 94C/30 sec at
57C/1 min at 72C, and finally for 3 min at 72C.
The PCR products were purified by using a Power
gel extraction Kit (TaKaRa Biosystems, Shiga,
Japan). A 409-bp fragment of the PCR product was
sequenced with an ABI3100 instrument (Applied
Biosystems 3100), and their identities as mtDNA
were confirmed by BLAST search.

RESULTS
A total of 289 in vivo matured oocytes from 41
females were obtained by flushing the oviduct
and a total of 251 reconstructed intrageneric embryos were transferred into 12 female dog recipients. Two pregnancies were detected by ultrasonography at 23 days of gestation in
recipient dogs. In each surrogate dog, the existence of two fetal sacs was confirmed by early
pregnancy diagnosis at 23 days (Fig. 1), but one
from each surrogate mother was delivered at
term (Fig. 1). One fetal sac from each pregnant
dog failed to maintain to term. The first cloned
wolf, named SNUWOLF for (Seoul National
University Wolf) was delivered by cesarean section on October 18, 2005, 60 days after embryo
transfer (ET). The birth weight was 430 g. The
second cloned wolf named SNUWOLFFY was
delivered on October 26, 2005, at 61 days postET, and its weight was 530 g. Both cubs display
gray wolf characteristics including distinctive
coarse gray coat color and razor-sharp teeth, not
dog phenotypes (Fig. 2).

FIG. 1. Ultrasonography images of pregnant recipient females. (A) Fetal vesicle (arrow) on ultrasonography on day
23 after embryo transfer (ET). (B) Fetal skull image (arrow) on day 34 after ET; diameter of fetal skull: 1.09 cm.

134

KIM ET AL.

FIG. 2. Cloned wolves by somatic cell nuclear transfer. (A) SNUWOLF, the first cloned wolf at 129 days after birth;
(B) SNUWOLFFY, second cloned wolf derived at 121 days after birth from a female gray wolf as a genetic donor (C).

To confirm the genetic identity of the cloned

wolves, microsatellite analysis was performed by
genomic DNA testing of the donor wolf (from
cultured nuclear donor fibroblasts), the two
cloned wolves (from a tail tissue fragment), and
the two surrogate female recipients (from blood
leukocytes). To identify microsatellite markers
that would distinguish the wolf donor from the
two surrogate female recipient dogs, a total of 27
canine markers were tested and 19 markers were
selected. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically
identical to the donor wolf (Table 1).
Sequence analysis also demonstrated that the
mitochondrial DNA (mtDNA) sequence of the
second cloned wolf was identical to that of the
oocyte donor (Table 2).

DISCUSSION
After Dolly, SCNT has been applied to the production of cloned livestock, and laboratory and
pet animals. Also, recently, it has been considered

to be a powerful tool for producing endangered

animals (Lanza et al., 2000; Loi et al., 2001; Gomez
et al., 2004). Accordingly, the present study applied SCNT to produce the endangered gray wolf
and resulted in the production of two live cloned
wolves. Our results showed the potential of
SCNT to recover the endangered or threatened
canids such as the red wolf, dingo, and fox.
Because of limitations in obtaining gray wolf
mature oocytes, in vivo matured canine oocytes
were used. After births of cloned puppies, procedures to obtain in vivo matured oocytes or manipulate the canine oocytes were stabilized and
improved. One of the improvements was an increase in fusion rates, reaching more than 87% in
cloned wolf embryos compared to 75% in cloned
dog embryos (Lee et al., 2005). Moreover, because
pregnancy was established only after embryo
transfer using early-stage nuclear transfer embryos in dog cloning, the activated interspecies
oocytes in the present study were also transferred
into the oviducts of recipient dogs less than 4 h
after oocyte activation. A total of 251 reconstructed interspecies embryos were transferred

135

ENDANGERED WOLVES CLONED FROM ADULT SOMATIC CELLS

TABLE 1.

ANALYSIS

OF

CANINE-SPECIFIC MICROSATELLITE LOCI

1st Cloned wolf

Donor cell

SNUWOLF

2nd Cloned wolf

Surrogate

SNUWOFFY

Surrogate

Marker

Peak1

Peak2

Peak1

Peak2

Peak1

Peak2

Peak1

Peak2

Peak1

Peak2

PEZ1
PEZ2
PEZ5
PEZ6
PEZ10
PEZ11
PEZ12
PEZ13
PEZ15
PEZ17
REN105L03
REN165M10
FH2140
FH2010
FH2054
CPH04
CPH07
CPH14
CPH22

114.1
118.0
101
171
237
123
278.8
221
226.9
199
230.5
198.5
180.1
228
155
145.1
173.4
122.5
110.3

118.1
126.0
105
173
278
125
281.5
225
253.0
203
237.0
198.5
194.0
228
155
147.3
173.4
131.2
112.4

114.2
118.0
101
171
237
123
278.9
221
227.1
199
230.6
198.6
180.3
228
155
145.1
173.4
122.5
110.3

118.1
126.0
105
173
278
125
281.6
225
253.1
203
237.0
198.6
193.3
228
155
147.3
173.4
131.3
112.3

118.1
130.6
109
171
291
136
270.9
173
213.7
207
241.2
198.6
178.1
223
163
140.8
169.4
131.2
110.2

118.1
130.6
109
173
291
136
301.6
229
222.5
215
241.2
20.8
189.9
235
167
140.8
175.6
131.2
110.2

114.2
118.1
101
171
237
123
279.0
221
227.1
199
230.6
198.6
180.2
228
155
145.1
173.4
122.5
110.3

118.2
126.0
105
173
278
125
281.6
225
253.2
203
237.1
198.6
194.0
228
155
147.3
173.4
131.4
112.4

114.2
126.0
105
181
264
129
288.8
216
209.3
207
234.9
200.7
178.0
224
151
140.9
175.7
147.9
108.2

114.2
130.0
109
184
264
129
294.2
221
218.7
211
237.0
200.7
187.9
228
167
143.0
177.7
152.1
110.3

Nineteen canine-specific markers4 were used for microsatellite assay. PEZ Markers are identified in U.S. patent
05874217; other markers are listed in http://research.nhgri.nih.gov/dog_genome/.
aSNUWOLF was born on October 18, 2005.
bSNUWOFFY was born on October 26, 2005.

into 12 female dog recipients. The efficiency of

gray wolf cloning in the present study, about
16.7% [2 wolves from 12 recipients; from 251 embryos (0.80%)], is improved compared to the percentage reported for the production of two cloned
dogs from 123 recipients [1.6%; from 1095 embryos (0.09%)], largely due to the use of conditions developed during our dog cloning studies
and due to better micromanipulation techniques
(e.g., increase of fusion rate) in the present study.
Twenty-seven canine microsatellite markers
were tested to determine the proper markers for
wolf. When testing markers, some markers
showed the same values between donor cells, surrogate recipient, or unrelated dogs (Canis familiaris) and wolf (Canis lupus). Thus, these markers
were excluded for the genotyping analysis. Microsatellite analysis with 19 markers confirmed
that two cloned wolves had a genotype identical
to the donor wolf.
Although two cloned wolves were consistent
with donor genotyping, in order to know mtDNA
distribution in those wolves, homoplasmy or heteromplasmy of mtDNA was investigated. Previously, intergeneric SNCT, homoplasmy mtDNA
was observed; mtDNA of cloned gaur (Lanza et

al., 2000) and mouflon (Loi et al., 2001) were exclusively derived from the recipient bovine and
ovine oocyte, respectively. However, in most
bovine SCNT studies, mtDNA heteroplasmy was
observed in cloned cattle (Steinborn et al., 2000;
Steinborn et al., 2002; Takeda et al., 2003). In the
present study, in line with the previous intergeneric studies in cloned gaur (Lanza et al., 2000),
mouflon (Loi et al., 2001), and dog (Lee and Park,
2006), mtDNA sequence of D-loop in the second
cloned wolf was identical to that of the oocyte
donor. Unfortunately, we could not compare
mtDNA sequence analysis between the first
cloned dog and its oocyte donor dog because we
failed to obtain the mtDNA from the first oocyte
donor dog because of the sudden death of the
donor dog.
In conclusion, this study demonstrated that
SCNT is a practical approach for conserving endangered canids.

ACKNOWLEDGMENTS
This study was supported by grants from the
Korean MOST (Top Scientist Fellowship) and

aGeneBank

T
T
C
T
T
T
T

A
A
A
G
A
A
A

access number: U96639.

Referencea
Donor cell
1st Cloned wolf
1st Surrogate mother
2nd Cloned wolf
2nd Oocyte donor dog
2nd Surrogate mother

MTDNA

SEQUENCES
OF

AN

OOCYTE DONOR DOG,

Nucleotide positionsa

DONOR CELL, TWO CLONED WOLVES,

AND

TWO SURROGATE MOTHERS

C
C
C
C
T
T
C

T
A
T
A
G
G
T

A
G
A
A
G
G
A

G
A
G
A
A
A
G

T
T
T
C
T
T
T

A
A
A
A
A
A
A

C
C
C
C
C
C
C

T
C
T
T
C
C
T

A
A
A
A
A
A
A

T
T
T
T
T
T
T

T
T
T
T
C
C
T

C
T
C
C
T
T
C

C
C
C
C
C
C
C

C
T
C
C
T
T
C

C
C
C
C
C
C
C

A
A
A
A
G
G
A

T
T
T
T
T
T
C

15625 15627 15659 1566 15670 15679 15827 15841 15726 15827 15838 15841 15842 15939 15969 15982 15986 16030 16052

TABLE 2.

ENDANGERED WOLVES CLONED FROM ADULT SOMATIC CELLS

KOSEF (grant # M10625030005-06N250300510). We

thank Seoul Grand Park Zoo for supporting the
donor cells and C. G. Park (KAIST) for assisting on
mtDNA sequencing analysis. The authors are
grateful for the graduate fellowship provided by
the Korean MOE, through the BK21 program.

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Address reprint requests to:

Dr. Byeong Chun Lee
Department of Theriogenology
College of Veterinary Medicine,
Seoul National University
Seoul 151-742, Korea
E-mail: bclee@snu.ac.kr