5241_e03_p247-258

9/30/04

9:34 AM

Page 247

CLONING AND STEM CELLS

Volume 6, Number 3, 2004
Mary Ann Liebert, Inc.

Birth of African Wildcat Cloned Kittens Born from

Domestic Cats
MARTHA C. GMEZ,1,2 C. EARLE POPE,1 ANGELICA GIRALDO,1,2 LESLIE A. LYONS,3
REBECCA F. HARRIS,1 AMY L. KING,1 ALEX COLE,1 ROBERT A. GODKE,2
and BETSY L. DRESSER1,4

ABSTRACT
In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell
donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell
nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single
AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two
sources of oocytes were compared, fusion rate was higher using in vivomatured oocytes as
recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitromatured
oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two
groups: recipients (n
24) receiving
25 embryos and recipients (n
26) receiving 30 embryos. Twelve recipients (46.2%) receiving 30 embryos were diagnosed to be pregnant, while
no pregnancies were established in recipients receiving
25 NT embryos. Also, to determine
the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer
on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of
embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n
17), versus day 5 (n
4) or day 6 (n
3) was significantly different. Of the 12 pregnant recipients,
nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%)
from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight
died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and
bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed
that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first
wild carnivores to be produced by nuclear transfer.
INTRODUCTION

HE SURVIVAL OF MOST SPECIES in the Felidae

family is considered as threatened or endan-

gered. In fact, 36 of 37 species of this family are

at risk of extinction. The population decline can
be attributed to poaching and human destruction
of natural ecosystems. As populations decrease,

1Audubon

Center for Research of Endangered Species, New Orleans, Louisiana 70131 USA.
of Animal Sciences, LSU Agricultural Center, Baton Rouge, Louisiana 70803 USA.
3School of Veterinary Medicine, University of California Davis, Davis, California 95616 USA.
4Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148 USA.
2Department

247

5241_e03_p247-258

9/30/04

9:34 AM

Page 248

248

insularization eventually results in subpopulation extinctions caused by detrimental genetic

and demographic effects (Nowell and Jackson,
1996a).
Domestic cats are a useful research model to
develop assisted reproductive technologies for
the conservation of endangered felids. During the
last two decades, the feasibility of oocyte recovery (in vivo maturation), in vitro maturation
(IVM), in vitro fertilization (IVF), in vitro embryo
culture (IVC) and embryo transfer (ET) to recipients has been demonstrated (Pope, 2000; Luvoni,
2000). The domestic cat can also serve as a successful recipient of embryos from closely related
small non-domestics cats as shown by the birth
of Indian desert cat (Pope et al., 1993) and African
Wildcat kittens (Pope et al., 2000) after transfer of
IVF-derived embryos.
Recently, the feasibility of producing viable domestic cat offspring by nuclear transfer was demonstrated (Shin et al., 2002). Regarding endangered felids, nuclear transfer is a potentially
valuable technique for assuring the continuation
of species with few remaining numbers of animals. In fact, cloning could maintain genetic variability in endangered felids when there are few
animals in a founder population by conserving
the maximum number of alleles for future breeding projects (Seidel, 2001).
Inter-species nuclear transfer involves the
transfer of a donor cell nucleus of one species into
an enucleated oocyte of another species. Since a
limited number of endangered felid oocytes are
available, the ooplasm of the domestic cat oocyte
can be used as the recipient cytoplast of a somatic
cell nucleus of an endangered cat. Recently, we
have shown that domestic cat ooplasm is a compatible host for somatic cell nuclei from an AWC
and the reconstructed embryos do undergo development in vitro (Gmez et al., 2003). Although
the feasibility of inter-species nuclear transfer has
been demonstrated only three live endangered
mammals have been produced: gaur (Lanza et al.,
2000), mouflon (Loi et al., 2001), and banteng
(Jansen et al., 2004) after transfer of NT embryos
into domestic ruminant recipients.
Incomplete nuclear reprogramming is a major
constraint to the in vivo developmental potential
of cloned embryos (Kikyo and Wolffe, 2000).
Most cloned blastocysts, regardless of species, are
not able to maintain fetal development to term
(Colman, 2000). Therefore, to maximize preg-

GMEZ ET AL.

nancy rates in species such as the pig, up to 62

reconstructed embryos are transferred into each
recipient (Polejaeva et al., 2000). In the domestic
cat, we reported that more pregnancies occurred
after uterine transfer of
12 IVF/IVC-derived
embryos (Pope et al., 1993). In vivo developmental potential of cloned embryos may be even more
susceptible to the stressful environment of in vitro
culture. Although in vitro development of cat embryos, as measured by blastocyst development
and number of cells per embryo, has improved
(Pope et al., 1999; Gmez et al., 2001, 2003), the
presence of perturbations affecting embryo viability such as nutritional imbalances, oxidative
stress, and defective genomic imprinting must be
acknowledged (Leese, 2002).
In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell donor
to evaluate the in vivo developmental competence, after transfer into domestic cat recipients,
of cloned embryos produced by the fusion of
AWC fibroblast cell nuclei with domestic cat cytoplasts. Specifically, our primary goals were to
(1) determine the minimal number of reconstructed embryos required per recipient to establish pregnancy; (2) evaluate the influence of in
vitro culture interval on establishment and maintenance of fetal development in recipients after
transfer of reconstructed embryos; and (3) evaluate factors affecting pre- and post-natal fetal survival and describe characteristics of the kittens.

MATERIALS AND METHODS

Animals
Domestic cats used as oocyte donors and embryo recipients were group-housed in environmentally controlled rooms with a 14-h/10-h
light/dark cycle at 2026C. The antibody defined cats were purchased from Liberty Research,
Inc. (Waverly, NY). The AWC male was housed
in a separate room under the same conditions.
The rooms were cleaned, and cats were fed once
daily (Science Diet, Hill Pet Nutrition). Fresh water was available at all times. All animal procedures were approved by the Institutional Animal
Care and Use Committee of the Audubon Center
for Research of Endangered Species as required
by the Health Research Extension Act of 1985
(Public Law 99-158).

5241_e03_p247-258

9/30/04

9:34 AM

Page 249

BIRTH OF AFRICAN WILDCAT CLONED KITTENS

Chemicals
Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise stated.

Establishment of adult AWC cell line

The cell line was generated from skin tissue collected by biopsy from a 3-year-old male AWC.
Collected tissue was finely cut into 1-mm2 pieces
and plated in a 75-cm2 tissue-culture flask (Nunc,
Denmark) containing 7 mL of Dulbecco Modified
Eagles Medium (DMEM) supplemented with 50

g/mL of gentamicin and 10% (v/v) fetal bovine
serum (FBS; Hyclone, Logan, UT) and cultured
at 38C in 5% CO2/air. After 710 days of incubation, monolayer outgrowths with fibroblasticlike morphology were disaggregated with 2.5
mg/mL of pronase, re-suspended in DMEM with
10% FBS and 10% (v/v) dimethyl sulfoxide, and
cooled at 1.0C/min to 80C (Mr. Frosty; Nalgene, Rochester, NY) before storage in liquid nitrogen (LN2). To prepare fibroblasts as donor cells
for NT, cells were thawed and cultured in DMEM
containing 10% FBS for 13 passages. Then, when
cells reached 80% confluency, FBS was reduced
to 0.5% and cells were cultured for an additional
5 days (serum starvation) before cryopreservation and storage in LN2.

Oocyte maturation
For in vitro maturation, cumulusoocyte complexes (COC) were recovered from ovaries of domestic cats within 25 h after ovariohysterectomy. Selected COCs were cultured in modified
TCM-199 containing 1 IU/mL hCG, 0.5 IU/mL
eCG, 10
g/mL epidermal growth factor, and 3
mg/mL BSA (Fraction V, fatty acid free; Serological Proteins, Kankakee, IL) for 24 h in 5% CO2,
5% O2 and 90% N2 at 38C (Gmez et al., 2003).
To obtain in vivomatured oocytes, domestic
cats were treated with a total of 34 IU of porcinefollicle stimulating hormone (FSH, Sioux Biochemical, Sioux Center, IA) given in decreasing
daily doses (s.c.) for 4 days, followed by 3 IU of
porcine-luteinizing hormone (im; LH, Sioux Biochemical) on day 5. At 2426 h after LH injection,
oocytes were collected by laparoscopic aspiration
of mature ovarian follicles (Gmez et al., 2000).
In vivomatured oocytes were used for NT immediately after aspiration.

249

Cumulus cells of in vivo and in vitromatured

oocytes were removed by vortexing in 1 mg/mL
of hyaluronidase (type IV) for 5 min, followed by
gentle mechanical pipetting. Denuded oocytes
were placed in Tyrodes solution (Irvine Scientific, Santa Ana, CA) with 1% MEM nonessential
amino acids, 3 mg/mL BSA, 15 mM NaHCO3,
0.36 mM pyruvate, 2.2 mM calcium lactate, 1 mM
glutamine, and 50
g/mL gentamicin (IVC-1
medium) at 38C in 5% CO2 until further use.

Nuclear transfer
The protocol for nuclear transfer has been described previously (Gmez et al., 2003). Briefly,
denuded metaphase II oocytes were incubated
for 15 min at 38C in Ca2-free and Mg2-free
modified Tyrodes salt solution supplemented
with 1% MEM nonessential amino acids, 3
mg/mL BSA, 30 mM NaHCO3, 0.36 mM pyruvate, 1 mM glutamine, 50
g/mL gentamicin
(ECM medium), 20
g/mL Hoechst 33342 and 20

g/mL cytochalasin B (CCB). After incubation,
oocytes were enucleated in ECM medium in
which supplemental NaHCO3 was reduced to 15
mM and to which 15 mM Hepes, 20
g/mL CCB
and 2 mg/mL sucrose were added. The first polar body and
10% of the underlying cytoplasm
were drawn into an enucleation pipette (20
m,
OD). Removal of the metaphase II plate was confirmed by brief exposure to epifluorescence microscopy.
A vial containing AWC fibroblast cells synchronized in G0/G1 phase by serum starvation
(Gmez et al., 2003) was thawed prior to being
used for NT, and a single AWC fibroblast cell was
introduced into the perivitelline space of each
enucleated oocyte. For fusion, each NT couplet,
in a solution of 0.3 M mannitol and 0.1 mM Mg2,
was placed between two stainless-steel electrodes
spaced 120
m apart (LF-101; Nepa Gene, Tokyo,
Japan) that were controlled by micromanipulators. Membrane fusion was induced by applying
a 3-sec AC pre-pulse of 20V, 1 MHz followed by
two 30-
sec DC pulses of 240V/mm at intervals
of 0.5 sec. Following the fusion pulses, couplets
were washed and cultured in IVC-1 medium supplemented with 7.8 mM calcium lactate (IVC-1 
Ca2) for 30 min., at which time fusion was evaluated visually by confirming the presence or absence of the donor cell in the perivitelline space.

5241_e03_p247-258

9/30/04

9:34 AM

Page 250

250

After 23 h of culture in IVC-1  Ca2, activation was induced by placing fused couplets between two electrodes in a fusion chamber containing 3 mL of a solution of 0.3 M mannitol, 0.1
mM Mg2, and 0.05 mM Ca2 and exposing them
to two 60-
sec DC pulses of 120 V/mm. Then,
couplets were incubated in 30-
L droplets of
IVC-1 medium supplemented with 10
g/mL cycloheximide and 5
g/mL CCB at 38C in 5% CO2
in air for 4 h.

Embryo culture
After activation, reconstructed embryos were
cultured in 500
L of IVC-1 medium in 5% CO2,
5% O2, and 90% N2 at 38C. Some couplets were
transferred to the oviducts of recipient females on
day 1. The remaining couplets were cultured until day 2 or 3, at which time, after uncleaved couplets were recorded and removed, embryos were
placed into 500
L of fresh IVC-1 medium containing 1% MEM essential amino acids (IVC-1 
EAA). Then, on day 5, the number of morulae
was recorded and all embryos were moved into
Tyrodes solution containing 1% NEAA, 2% EAA,
10% FBS, and the same supplements as in IVC-1
medium (IVC-2 medium) and cultured until
transfer to a recipient.

Embryo transfer
Cloned embryos were transferred into the
oviducts or uteri of 50 gonadotropin-treated domestic short hair (DSH) recipients. Day 1 embryos were transferred to the oviducts of recipients, some of which had served as oocyte donors
on day 0 (n  2) and others of which served as
recipients only after induction of ovulation (n 
14) using a lower dose of FSH (13 IU) and a
higher dose of LH (5 IU) than the oocyte donors
received. For oviductal embryo transfer, the abdominal cavity was visualized via a 5-mm endoscope inserted just above the umbilicus, and a 16
gauge  6.35 cm stainless steel trocar/cannula
was inserted transabdominally near the midline

5 cm below the umbilicus. Then, a 14.5-cm tom
cat catheter (3.5 French, Sherwood Medical, St.
Louis, MO) was passed through the 16-gauge
cannula and directed through the infundibulum
overlaying the ovary and into the upper ampullary portion of the oviduct. A 50-cm length of
sterile polyethylene tubing (PE 10, no. 427400,
Becton Dickinson, Sparks, MD) containing the

GMEZ ET AL.

embryos near the lower tip was threaded through

the tom cat catheter, and the embryos were expelled into the oviduct using positive pressure
from a 1-mL syringe.
The recipients of day 5, 6, and 7 embryos were
gonadotropin-treated females that had undergone laparoscopic oocyte aspiration 57 days previously. Briefly, one uterine horn was exteriorized through a 1.5-cm mid-ventral incision and
punctured with a sterile 16-gauge round-tipped,
short bevel trocar
1 cm from the anterior tip.
Embryos were aspirated in
50
L of IVC-2
medium into a 14.5-cm tom cat catheter using a
1-mL plastic syringe. The catheter was then
threaded 45 cm into the uterine lumen before depositing the embryos (Pope et al., 1994).

Pregnancy detection and parturition

Each recipient was checked for pregnancy status using abdominal ultrasonography on day
2123 after ovulation or oocyte aspiration. Pregnant recipients were monitored by ultrasonography on a weekly basis until day 60, at which
time daily ultrasonographic examinations were
made to check for fetal movement and heart beat.
Pregnant recipients were allowed to complete
gestation, at which time kittens were delivered
by Caesarean section, either (1) as an intervention
after onset of vaginal bleeding and/or occurrence
of labor symptoms or (2) on a pre-scheduled day
before symptoms of parturition were seen. In an
effort to reduce the onset of premature parturition, some pregnant cats (n  3) were orally administered altrenogest (0.08 mg/kg, Regu-Mate,
Hoechst; Kustritz, 2001) daily from days 55 to
6567 of gestation. Also, in an attempt to reduce
perinatal kitten mortality (n  7) cats were
treated with four injections (im), 12 h apart, of
betamethasone (0.1 mg/kg, CelestoneSoluspan,
Schering) starting at varying times after day 60 of
gestation.

Microsatellite analysis
To determine the clonal status of all kittens,
DNA was extracted using standard phenol/chloroform techniques from the fibroblast cells and
blood of the AWC donor, blood of the recipient
dam, umbilical cord blood of live kittens, or
spleen of dead kittens. Standard sodium hydroxide isolation was used to isolate DNA from cheek
swabs of live kittens. Feline derived microsatel-

5241_e03_p247-258

9/30/04

9:34 AM

Page 251

251

BIRTH OF AFRICAN WILDCAT CLONED KITTENS

lite loci were amplified as previously reported by

Raymond et al. (1999) using standard ABI fluorescent chemistries on an ABI 377. The results
were analyzed using the software STRand
(Hughes).

Statistical analyses
The Chi square test was used to test the differences between treatments. Statistical difference was set at the p  0.05 level.

RESULTS
Embryo production after NT
A total of 2,565 in vivo (n  1751) and in vitro
(n  814) matured domestic cat oocytes were mechanically enucleated and electrically fused to
frozen-thawed AWC fibroblasts. Fusion efficiency was higher when in vivomatured oocytes
(1696/1751; 96.8%) were used as recipient cytoplasts as compared with in vitromatured oocytes
(736/814, 90.4%, p  0.05). In contrast, cleavage
rate of embryos reconstructed with in vitromatured oocytes (435/511, 85.1%) was higher than
that of embryos reconstructed with in vivomatured oocytes (944/1194, 79.1%, p  0.05). Some
reconstructed couplets (n  727) were not included in the cleavage rate evaluation because
they were transferred to domestic cat recipients
on day 1 before cleavage occurred.

consistently establish pregnancies, AWC cloned

embryos were transferred into domestic cat recipients within two groups: (1) recipients (n  24)
that received fewer than 25 embryos (mean 
19.4; range  825), and (2) recipients (n  26)
that received more than 30 embryos (mean 
41.7; range  3159). Also, to determine the influence of length of in vitro culture on pregnancy
rate we compared the transfer of day 1 embryos
into the oviduct (n  588) with transfer of day 5
(n  467), day 6 (n  355), and day 7 (n  142)
embryos into the uterus. Embryos were at the
12-cell stage on day 1, morulae on day 5 and day
6, and morulae and blastocysts on day 7 of culture.
Twelve of 26 (46.2%) recipient cats receiving
more than 30 NT embryos were diagnosed to be
pregnant when examined by ultrasonography at
2123 days after induction of ovulation or aspiration of follicular oocytes. In contrast, none of
the recipients receiving less than 25 embryos (n 
24) were diagnosed pregnant (Table 1). Although,
no differences were observed in pregnancy rates
after transfer of
30 embryos on day 1 (6/12,
50.0%), day 5 (4/9, 44.4%), or day 6 (2/5, 40.0%)
to synchronous recipients, a significant difference
was found in the number of fetuses implanted after transfer embryos on day 1 (n  17) versus day
5 (n  4) or day 6 (n  3; p  0.05; Table 1). Six of
7 (85.7%) pregnant cats with multiple fetuses
were recipients of day 1 embryos.

Embryo transfer and pregnancies

Birth of AWC cloned kittens and

histological analysis

To determine the number of reconstructed embryos that should be transferred per recipient to

Of the 12 pregnant recipients, nine (75%) pregnancies developed to term (60 days). In the

TABLE 1.

EFFECTS

OF

TRANSFER DAY AND NUMBER OF EMBRYOS ON OCCURRENCE OF PREGNANCY

RECIPIENTS RECEIVING AFRICAN WILDCAT (AWC) CLONED EMBRYOS

IN

DOMESTIC CAT

AWC NT embryos
Average
per recipient
Embryos per recipient
25

30
Total

ET, day

Total, n

7
6
5
1
6
5
1

142
172
50
102
183
417
486
1552

Recipients

n  SD

Total, n









9
9
2
4
5
9
12
50

15.7
19.1
25.0
25.5
36.6
46.3
40.5

5.5
4.4
0
0.5
4.1
12.1
10.5

Pregnant, n (%)
0
0
0
0
2
4
6
12

(0)
(0)
(0)
(0)
(40.0)
(44.4)
(50.0)
(24.0)

Fetuses, n

3
4
17
24

5241_e03_p247-258

9/30/04

9:34 AM

Page 252

252

GMEZ ET AL.

three (25%) other pregnancies, a total of five fetuses were present at day 2123, four of which
were reabsorbed between days 30 and 40, and one
which was spontaneously aborted on day 48 of
gestation (Table 2).
Six of the nine term pregnancies, consisting of
a total of eight fetuses, were not hormonally
treated during the last 710 days of pregnancy
(Table 2). Two of these recipients each had a single kitten that died in uteri within 24 h of delivery by Caesarean section on days 64 and 67 of
gestation. Histological analysis of the two kittens
showed no indications of infectious disease. No
inflammatory or other degenerative lesions were
recognizable in the tissue sections and all organs
appeared to be developing normally. One of the
two kittens had moderate haemorrhage within
the choroids plexus of the brain, possibly due to
hypoxia that occurred after placental separation.
The other four recipients delivered six kittens
by Caesarean section on days 6267 of pregnancy
after vaginal bleeding occurred. Of the six kittens,

four experienced respiratory failure shortly after

delivery and resuscitation efforts were not successful, one kitten lived for 36 h and one kitten
had died in utero at
45 days of gestation.
Analysis of the kitten that died 36 h after delivery showed multifocal moderate to severe
acute ulcerative enterocolitis associated with rodshaped bacteria, and squames were present
within the alveolar spaces of the lungs. These results suggest that the kitten had complications
caused by bacterial septicemia of clostridial origin.
The kitten that died in utero on
45 days of
gestation showed gross physical abnormalities,
including incomplete closure of the ventral body
wall musculature with abdominal organ exteriorization, severe cerebella and cerebellum aplasia and no recognizable central nervous system.
All the other kittens that died shortly after delivery had a normal physical appearance except
for one that showed incomplete closure of the
ventral body wall musculature with abdominal

TABLE 2. IN VIVO DEVELOPMENT OF AFRICAN WILDCAT (AWC)

CLONED EMBRYOS AFTER OVIDUCTAL OR UTERINE TRANSFER
Pregnant
recipients

AWC kittens (days 4870)

Total
fetuses
days
2123

Fetuses
reabsorbed
days
3040

Delivery
day

ET
day

Embryos
transferred
n

1
2
3
4
5
6a
7a

5
1
1
6
5
1
6

41
42
56
39
35
59
42

1
2
2
1
1
4
2

1
2
1

48
64
67
62
62

8a
9a

5
1

43
34

1
2

62
67

10b
11b

5
1

39
40

1
5

69
65

Stillborn

1
1c
1c

1d,e

1c
1d
12b

Total
aPregnant

35

24

67

cats showed vaginal bleeding.

cats treated with progestagen and betamethasone.
c Died in uteri just before delivery.
dDied in utero on day 4550 of gestation.
eKittens with body wall defect.
fKittens died within 3672 h after birth.
gDied at 6 weeks of age.
bPregnant

1c
7

Live 36 h

Healthy

1e
1
1f
1
1
1
1
1.g
1e
1f
7

Weight, g

90.4
108.7
60.5
95.4
102.2
77.0
107.0
50.0
98.3
92.5
73.0
86.9

71.9
111.0
105.2
88.7

5241_e03_p247-258

9/30/04

9:34 AM

Page 253

BIRTH OF AFRICAN WILDCAT CLONED KITTENS

organ exteriorization. Histological evaluation of

the lungs of the kittens revealed variable collapse
of alveolar spaces and presence of epithelial
squames within the lumen of alveolar spaces, indicating that alveoli were not inflated and the
lungs were immature. Thus, prenatal and early
neonatal mortality appears to have been primarily due to lung immaturity resulting from the
spontaneous initiation of premature parturition.
Three pregnant cats that were hormonally
treated during the last 1012 days of gestation delivered eight kittens by Caesarean section. The
four kittens that survived were born on 6 August
2003, 15 November 2003, and 12 January 2004 at
69, 65, and 67 days of gestation, respectively
(Table 2).
Of the four kittens that did not survive, two
died in utero within hours of delivery on days 64
and 66. One kitten that died within a few minutes after delivery had incomplete closure of the
ventral body wall musculature with abdominal
organ exteriorization. Another kitten died in utero
on
50 days of gestation and was being resorbed
at delivery.
Pathology analysis of one kitten that died in
utero on day 64 revealed diffuse adrenal congestion and mild nephrosis. Necrosis of the renal
tubular epithelium could be induced by endotoxic shock. The hypoxia may have been caused
by cardiovascular collapse and release of nephrotoxins before death. Therefore, prenatal death
was possibly due to hypoxia that occurred after
placenta separation and is probably analogous to
the prenatal death occurring in pregnant recipi-

253

ents that were not hormonally treated during the

final stage of gestation.
Two kittens, currently 7 and 3 months of age
(Fig. 1) are healthy and developing normally. Of
the other two kittens, one died at 56 h after birth
and one died at 6 weeks of age. Histological analysis of the kitten that died at 56 h after birth indicated a diffuse moderate septic and supurative
pneumonia with abnormally high bacterial
counts in several organs. The bacteria may have
gained entry through the urachus and extended
into the urinary bladder.
The kitten that died at 6 weeks of age had a
congenital megaesophagus and acute supurative
pneumonia. The primary cause of death was
pneumonia, likely a result of aspiration of formula during bottle-feeding.
As shown in Table 2, the birth average weight
of live kittens (92.3 g; range  71.9105.0 g) was
similar to the average weight of kittens that died
during the peri- and post-parturient period (87.4
g; range  50.0111.0 g). Phenotypically, all
cloned kittens (n  17) were African Wildcats.
Subsequent DNA analysis of 12 cat-specific microsatellite loci performed with blind testing at
the School of Veterinary Medicine, University of
California, Davis, confirmed that they were identical to DNA of the AWC donor male (Jazz;
Table 3).

Placental analysis
A total of 11 placentae were analyzed. Macroscopic analyses showed that five placentae had

FIG. 1. African wildcat cloned kittens (A) Ditteaux born on 6th August 2003, at two months after birth. (B) Miles
and Otis born on 15th November 2003, at seven days after birth.

5241_e03_p247-258

9/30/04

9:34 AM

Page 254

254

GMEZ ET AL.
TABLE 3.

ANALYSIS

OF

FELINE GENETIC MARKERS

AWC donor
Jazz

AWC cloned kittens

(n  17)

Feline
markers

Fibroblasts

Blood

Cheek
cells

Umbilical
cord

FCA075
FCA097
FCA201
FCA224
FCA229
FCA293
FCA305
FCA441
FCA018
FCA145
FCA651
FCA674

132/134
140/146
153/155
160/178
158/162
179/187
191/201
158/166
233/233
174/174
135/135
148/148

132/134
140/146
153/155
160/178
158/162
179/187
191/201
158/166
233/233
174/174
135/135
148/148

132/134
140/146
153/155
160/178
158/162
179/187
191/201
158/166
233/233
174/174
135/135
148/148

132/134
140/146
153/155
160/178
158/162
179/187
191/201
158/166
233/233
174/174
135/135
148/148

Values represent the sizes of the two alleles of the

amplified microsatellites markers.

patches of serous-lacy tissue replacing normal

placental tissue. Microscopic analysis of these five
placentae showed that areas of the placental epithelium were reduced in thickness and had
shorter and thicker papillary projections or
merely a thin band of edematous fibrous connective tissue. These observations indicate the
ocurrence of multifocal placental atrophy with fibrous tissue replacement. Curiously, these abnormal placentae allowed development of kittens
to term, including two kittens that survived. The
placenta of a kitten that died in uteri before delivery by Caesarean section showed excessive abnormal mineralization and intermittent serosanguineous exudates. These abnormalities were not
present in all the placental sections analyzed;
some sections were histologically normal.

DISCUSSION
In the present study, we demonstrated that
AWC kittens can be produced after transfer of
embryos derived by fusion of adult somatic cells
from one species with enucleated oocytes of a
closely related species (domestic cat). We have
shown that, under our current laboratory conditions, transfer of at least 30 NT embryos per recipient was required for establishing a pregnancy. Also, the length of in vitro culture affected
the number of embryos that successfully underwent fetal development. The primary causes of
peri-natal mortality were placental separation,

lung immaturity at premature delivery and bacterial septicemia.

We obtained pregnancies only in recipients
that received
30 NT embryos. A possible explanation for the high number of embryos required for producing pregnancies is that most of
the reconstructed embryos had inadequate nuclear remodeling or incomplete reprogramming
(Gmez et al., 2003). We previously demonstrated
in AWC reconstructed embryos that 18% were
fragmented and unable to undergo chromatin remodeling, 38% arrested at the 810-cell stages and
44% and 24% reached the morula and blastocysts
stages, respectively (Gmez et al., 2003). In this
study, NT embryos were reconstructed from the
same AWC somatic cell line and oocytes were
similar in type and source to those used in previous NT experiments. Then, from the mean
number of embryos transferred on day 1 (40.5),
day 5 (46.3), and day 6 (36.6), we can estimate that
at least 10 embryos should develop to the blastocyst stage. Based on these values, we suggest that,
using our current methods, to consistently establish pregnancies in domestic cats receiving AWC
NT-derived embryos, at least 30 embryos should
be transferred per recipient. If it is assumed that
NT-derived cat blastocysts may be less developmentally competent than IVF-derived cat blastocysts, these numbers directly correlate with the
minimal number of IVF derived embryos required to reliably establish pregnancies in domestic cat recipients.
Domestic cat recipients receiving day-7 blastocysts (mean  7.4) and morulae (mean  8.3) on
day 7 post-oocyte retrieval did not establish pregnancies. During this portion of the study we were
transferring an average of 10 blastocysts per cat.
We do not think that the longer in vitro culture
interval was the primary reason for the developmental failure because we have produced domestic cat kittens after transfer of either 10 or 11
IVF-derived day-7 blastocysts per recipient (Pope
et al., 2003). Further experiments should determine if pregnancies can be established after transferring AWC cloned blastocysts.
In sheep, more lambs were produced after
early transfer (day 2) of IVF embryos as compared
to later transfer at day 6 (Walmsley et al., 2004).
Similarly, in the present study, a higher percentage of NT embryos developed into fetuses when
the transfer was done after a shorter period of culture (day 1, n  17/486, 3.5%) as compared with
embryos that were cultured for longer periods

5241_e03_p247-258

9/30/04

9:34 AM

Page 255

BIRTH OF AFRICAN WILDCAT CLONED KITTENS

(day 5, n  4/417, 1.0%; day 6, n  3/183, 1.6%).

From these results, we suggest that the developmental potential of some NT embryos is rescued by transferring them to recipient cats as uncleaved couplets and two-cell embryos; thereby
minimizing the detrimental effects of in vitro culture (Leese, 2002). Although more embryos implanted after transfer on day 1, fetal mortality by
day 48 of gestation also increased (6/17; 35.3%)
as compared with embryos transferred on day 5
(1/4; 25.0%) or day 6 (0/3; 0%). Also, three kittens that were born after embryo transfer on day
1 had incomplete closure of the ventral body wall
musculature with abdominal organ exteriorization. Because few fetuses were evaluated, we cannot conclude that the increase in fetal mortality
or the physical abnormalities were directly related to the shorter period of culture. However,
we can speculate that losses occurring after transfer on day 1 are not found after the transfer of
embryos on day 5 or 6 because the developmental potential of such embryos is sufficiently reduced by the extended in vitro culture interval
that they are incapable of further development in
utero.
Although embryo viability was demonstrated,
embryo survival after transfer was quite modest
(1.03.5%) and was similar to the overall efficiency of cloning reported in other species
(1.04.0%; Wilmut and Paterson, 2003). In addition to incomplete reprogramming of the differentiated nucleus, another factor that may contribute to low cloning efficiency is variation in
oocyte quality. Both in vivomatured and in
vitromatured oocytes have been used as recipient cytoplasts for production of cloned animals.
For example, in sheep, higher blastocyst development rates, pregnancy rates and embryo
survival were reported when in vivomatured
oocytes were used as donor cytoplasts (Wells et
al., 1997). In contrast, in goats no differences were
found in fusion rates, embryo development and
in the number of cloned kids produced from
oocytes harvested from FSH-stimulated donor
animals or from in vitromatured oocytes recovered from abbatoir ovaries (Reggio et al., 2001).
In pigs, in vivomatured oocytes have been used
extensively as cytoplast recipients (Miyoshi et al.,
2003). Walker et al. (2002) reported that in
vitromatured pig oocytes could also be used to
produce cloned piglets; however, a direct comparison between the two sources of cytoplasts has
not been done. In the present study, we found

255

higher fusion rates with in vivomatured cytoplasts and higher cleavage rates with in vitromatured cytoplasts. In a previous study, we found
no differences in blastocyst development in vitro
when in vivomatured and in vitromatured cat
oocytes were used as recipient cytoplasts of AWC
cells (Gmez et al., 2003). A primary purpose of
the present study was to find out if AWC kittens
could be produced by transfer of NT embryos to
domestic cat recipients. Because we were unable
to produce NT pregnancies after transferring
17 embryos per recipient in a previous study
(Gmez et al., 2003), in the present study in order to transfer
25 embryos per recipient, for
most transfers it was necessary to combine reconstructed embryos derived from both in
vitromatured and in vivomatured domestic cat
cytoplasts. The one domestic cat recipient receiving NT embryos derived from in vivomatured
cytoplasts did establish pregnancy and produce
a live kitten. We do not know if the other NT kittens originated from embryos derived from in
vivomatured or in vitromatured cytoplasts. Certainly, the comparative developmental competence of in vitromatured versus in vivomatured
cat cytoplasts requires investigation.
Several developmental abnormalities occur in
pregnancies from somatic cell nuclear transferderived embryos, including a high rate of
abortion during early gestation and a number of
perinatal complications. One complication we encountered in some cats was the occurrence of
vaginal bleeding by day 6267 of pregnancy without overt signs of labor. Although the kittens
were delivered within the normal gestation period of domestic cats (6271 days; Herron, 1977)
and African Wildcats (5663 days; Nowell and
Jackson, 1996b), the kittens died shortly after delivery from respiratory failure, a condition usually associated with premature delivery.
In domestic cats, serum progesterone levels begin a slow decline by day 50 of pregnancy, progressing to a precipitous decrease during the last
week prior to parturition (Verhage et al., 1976).
Although we did not measure serum progesterone levels in pregnant recipients, it seems possible that, since the recipient domestic cats were
gestating AWC cloned fetuses, feto-placental endocrine signaling may have been inadequate. As
a result, progesterone levels may have shown an
abnormally early decrease, starting a cascade of
pre-parturient events that resulted in vaginal
bleeding. Consequently, with the last three preg-

5241_e03_p247-258

9/30/04

9:34 AM

Page 256

256

nant recipients, in an effort to reduce perinatal

kitten mortality, a synthetic progestagen (altrenogest) was orally administered. Altrenogest
has been shown to maintain gestation in ovariectomized mares and dogs (Hinrichs et al., 1986;
Eilts et al., 1994). Since pregnancy was successfully maintained (until elective Caesarean section) in cats treated with the synthetic progestagen, luteal insufficiency may be implicated in the
occurrence of premature vaginal bleeding. Maternal administration of betamethasone is widely
used to improve fetal lung maturation (Senat,
2002; Celik et al., 2002). Recipients administered
both synthetic progestagen and betamethasone
during the latter part of gestation produced a total of five live kittens. Thus, while the improvement in fetal survival rate cannot be conclusively
attributed to the treatment combination of progestagen and betamethasone, the present results
do provide at least circumstantial evidence of
their effectiveness.
Placental abnormalities are a major cause of
mortality in cloned animals and likely contribute
to a number of perinatal complications in farm
animals (Cibelli et al., 2002). Abnormal placentae
have been reported in cloned mice, cattle and
sheep (Shimozawa et al., 2003; Hill et al., 2000;
Hartwich et al., 2003) and may be a result of aberrant gene expression (Suemizu et al., 2003). In our
study, histological analysis of placental tissue revealed a reduction in thickness and multifocal atrophy. In fact, some kittens died from hypoxia after placental separation, with respiratory failure
as a secondary consequence. Accordingly, it is
plausible that placental anomalies/dysfunction
were either directly or indirectly responsible for
the perinatal deaths of the cloned kittens. To improve success rates and reduce neonatal deaths,
basic studies are needed to identify factors causing abnormal placentation.
Developmental anomalies occur with a greater
frequency in cloned animals than in the average
population. Diagnostic pathology of cloned
lambs that died after birth revealed several
cloning-related abnormalities, some of which
were analogous to congenital human diseases
(Rhind et al., 2003). For example, four of eight
cloned lambs exhibited fetal overgrowth and a
body wall defect. These abnormalities are linked
to Beckwith-Wiedemann syndrome (B-WS), a human genetic syndrome characterized by abdominal wall defects, visceromegaly, macroglossia,
pre- and postnatal overgrowth and neonatal

GMEZ ET AL.

hypoglycemia. This disorder involves the deregulation of several genes, including insulin-like
growth factor, type 2 (IGF2) and cyclin-dependent kinase inhibitor 1C (CDKN1C; Murrel et al.,
2004). The product of IGF2 is a major fetal growth
factor, and is associated with overgrowth in fetuses derived from sheep embryos cultured in
vitro before transfer to recipients (Young et al.,
2001). Recently, in humans, an association was
found between babies produced by in vitro technology and the incidence of B-WS (DeBaun et al.,
2003). In our study, a major abnormality found in
three cloned kittens was incomplete closure of
ventral body wall musculature with exteriorization of abdominal organs; however, pathology associated with overgrowth was not observed.
Likewise, knockout mice for CDKN1C have been
found to manifest an anterior wall defect but do
not exhibit fetal overgrowth or other features of
B-WS (Fitzpatrick et al., 2002). Whether body wall
pathology is caused by gene deregulation or is
merely a consequence of another wall anomaly is
not known.
The influence of possible mitochondrial heteroplasmy and/or a deleterious mtDNA effect on
neonatal death of the cloned AWC kittens cannot
be discounted. Indeed, in future studies the precise mitochondrial inheritance pattern in AWC
cloned kittens will be evaluated to determine its
influence on survival rate.
The effectiveness of our cloning procedure was
shown by the birth of healthy AWC kittens. These
births represent the first wild carnivores to be
produced by NT and further expand the growing list of species in which cloned offspring have
been produced. Our success may be attributable
to improvements in the embryo culture system
(Gmez et al., 2003), refinements made to the nuclear transfer procedure (Gmez et al., 2003), increasing the number of embryos transferred, reducing the culture interval before transfer and
modification of current protocols successfully
used in other species to manage pregnancy and
improve viability of offspring (Ptak et al., 2002).
While there are major limitations to current
cloning technologies, knowledge about nuclear
transfer procedures is advancing rapidly and
technology is improving. Researchers should be
accessible to the possibilities offered by nuclear
transfer as one of the assisted reproduction techniques potentially useful for the conservation of
endangered species, which in some cases, may offer the prospect of species continuation rather

5241_e03_p247-258

9/30/04

9:34 AM

Page 257

BIRTH OF AFRICAN WILDCAT CLONED KITTENS

than extinction. Future research should focus on

defining and understanding the mechanisms responsible for the abnormalities, so that efficiency
is improved and neonatal death rate reduced. We
expect that advances in understanding the molecular events for reprogramming of the donor
genome will contribute to clarifying the derivation of developmental defects in cloned embryos.

ACKNOWLEDGMENTS
We are grateful to Stella Sullivan for her excellent cat care and hand rearing some of the
cloned kittens. In addition, we thank Dr. Karen
Miller, Dr. Steve Miller, Barbara Vincent and the
Audubon Nature Institute veterinary staff for
their help with the surgery procedures; Jackie
Coulon for coordinating and collecting ovaries
from the veterinary clinics; and the staff and interns from the Species Survival Center for the 24
h per day cat observations towards the end of
each pregnancy.

REFERENCES
Celik, C., Acar, A., Cicek, N., et al. (2002). Corticosteroid
treatment for prevention of prematurity complications.
Arch. Gynecol. Obstet. 267, 9094.
Cibelli, J.B., Campbell, K.H., Seidel, G.E., et al. (2002). The
health profile of cloned animals. Nat. Biotechnol. 20,
1314.
Colman, A. (2000). Somatic cell nuclear transfer in mammals: progress and applications. Cloning 1, 185200.
Debaun, M.R., Niemitz, E.L., and Feinberg, A.P. (2003).
Association of in vitro fertilization with BeckwithWiedemann syndrome and epigenetic alterations of
LIT1 and H19. Am. J. Hum. Genet. 72, 156160.
Eilts, B.E., Paccamonti, D.L., Hosgood, G., et al. (1994).
The use of Ally-trenbolone as a progestational agent to
maintain pregnancy in ovariectomized bitches. Theriogenology 24, 12371245.
Fitzpatrick, G.V., Soloway, P.D., and Higgins, M.J. (2002).
Regional loss of imprinting and growth deficiency in
mice with a targeted deletion of KvDMR1. Nat. Genet.
32, 426431.
Gmez, M.C., Pope, C.E., Harris, R.F., et al. (2000). Births
of kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro. Reprod.
Fertil. Dev. 12, 423433.
Gmez, M.C., Jenkins, J.A., Giraldo, A., et al. (2003). Nuclear transfer of synchronized African wildcat somatic
cells into enucleated domestic cat oocytes. Biol. Reprod.
69, 10321041.
Gmez, M.C., Pope, C.E., Harris, R.F., et al. (2003). De-

257

velopment of in vitro matured, in vitro fertilized domestic cat embryos following cryopreservation, culture
and transfer. Theriogenology 60, 239251.
Hartwich, K.M., Walker, S.K., Jurisevic, A.M., et al. (2003).
Evidence of impaired placental function in cloned fetal
sheep during late gestation. Theriogenology 59, 256 [abstr].
Herron, M.A. (1977). Feline reproduction. Vet. Clin. North
Am. 7, 715722.
Hill, J.R., Burghardt, R.C., Jones, K., et al. (2000). Evidence
for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses. Biol. Reprod. 63, 17871794.
Hinrichs, K., Sertich, P.L., and Kenny R.M. (1986). Use of
altrenogest to prepare ovariectomized mares as embryo
transfer recipients. Theriogenology 26, 455460.
Janssen, D.L., Edwards, M.L., Koster, J.A., et al. (2004).
Postnatal management of chryptordich banteng calves
cloned by nuclear transfer utilizing frozen fibroblast
cultures and enucleated cow ova. Reprod. Fertil. Dev.
16, 224(abst).
Kikyo, N., and Wolffe, A.P. (2000). Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons. J. Cell Sci. 113, 1120.
Kustritz, R. (2001). Use of supplemental progesterone in
management of canine pregnancy. In: Recent Advances
in Small Animal Reproduction. P.W. Concannon, G. England, and J. Verstegen, eds. (Ithaca: International Veterinary Information Service), p. A1220.0401.
Lanza, R.P., Cibelli, J.B., Diaz, F., et al. (2000). Cloning of
an endangered species (Bos gaurus) using interspecies
nuclear transfer. Cloning 2, 7990.
Leese, H.J. (2002). Quiet please, do not disturb: a hypothesis of embryo metabolism and viability. BioEssay
24, 845849.
Loi, P., Ptak, G., Fulka, J., Jr., et al. (2001). Genetic rescue
of an endangered mammal by cross-species nuclear
transfer using post-mortem somatic cells. Nat. Biotechnol. 19, 962964.
Luvoni, G.C. (2000). Current progress on assisted reproduction in dog and cats: in vitro embryo production.
Reprod. Nutr. Dev. 40, 505512.
Miyoshi, K., Rzucidlo, S.J., Pratt, S.L., et al. (2003). Improvements in cloning efficiencies may be possible by
increasing uniformity in recipient oocyte and donor
cells. Biol. Reprod. 68, 10791086.
Murrell, A., Heeson, S., Cooper, W.N., et al. (2004). An association between variants in the IGF2 gene and Beckwith Wiedemann syndrome: interaction between genotype and epygenotype. Hum. Mol. Genet. 13, 247255.
Nowell, P., and Jackson, P. (1996a). Cats and habitat loss.
In Status Survey and Conservation Action Plan: Wild Cats
(Gland, Switzerland, IUCN), pp. 149179.
Nowell, K., and Jackson, P. (1996b). African wildcat
group. In Status Survey and Conservation Action Plan:
Wild Cats (Gland, Switzerland, IUCN), pp. 3235.
Polejaeva, I.A., Chen, S.H., Vaught, T.D., et al. (2000).
Cloned pigs produced by nuclear transfer from adult
somatic cells. Nature 407, 8690.
Polge, C., Rowson, L.E., and Chang, M.C. (1966). The ef-

5241_e03_p247-258

9/30/04

9:34 AM

Page 258

258
fect of reducing the number of embryos during early
stages of gestation on the maintenance of pregnancy in
the pig. J. Reprod. Fertil. 12, 395397.
Pope, C.E. (2000). Embryo technology in conservation efforts for endangered felids. Theriogenology 53, 163174.
Pope, C.E., Keller, G.L., and Dresser, B.L. (1993). In vitro
fertilization in domestic and non-domestic cats including sequences of early nuclear events, development in
vitro, cryopreservation and successful intra- and interspecies embryo transfer. J. Reprod. Fertil. 47, 189201.
Pope, C.E., McRae, M.A., Plair, B.L., et al. (1994). Successful in vitro and in vivo development of in vitro fertilized two- to four-cell cat embryos following cryopreservation, culture and transfer. Theriogenology 42,
51325.
Pope, C.E., Schmid, R., and Dresser, B.L. (1999). In vitro
development of cat embryos produced by in vitro fertilization is enhanced by addition of cysteine to the maturation medium and a reduced O2 atmosphere. Theriogenology 51, 291(abst).
Pope, C.E., Gmez, M.C., Mikota, S.K., et al. (2000). Development of in vitroproduced African Wildcat (Felis
silvestris) embryos after cryopreservation and transfer
into domestic cat recipients. Biol. Reprod. 62, 321(abst).
Pope, C.E., Gmez, M.C., King, A.L., et al. (2003). Embryos produced in vitro after recovery of oocytes from
cat ovaries stored at 4C for 24 to 48 hours retain the
competence to develop into live kittens after transfer to
recipients. Theriogenology 59, 308(abst).
Ptak, G., Clinton, M., Tischner, M., et al. (2002). Improving delivery and offspring viability of in vitroproduced
and cloned sheep embryos. Biol. Reprod. 67, 17191725.
Raymond, M.M., David, V.A., Lyons, L.A., et al. (1999).
A genetic linkage map of microsatellites in the domestic cat (Felis catus). Genomics 57, 923.
Rhind, S.M., King, T.J., Harkness, L.M., et al. (2003).
Cloned lambslessons from pathology. Nat. Biotechnol. 21, 744745.
Seidel, G.E., Jr. (2001). Cloning, transgenesis, and genetic
variance in animals. Cloning Stem Cells 3, 251256.
Senat, M.V. (2002). Corticosteroid for fetal lung maturation: indication and treatment protocols. J. Gynecol. Obstet. Biol. Reprod. 31, 672676.

GMEZ ET AL.
Shimozawa, N., Tajima, S., Azuma, N., et al. (2003). Histological study of the hypertrophic placentas and open
eyelids observed in cloned fetuses. J. Reprod. Dev. 49,
221226.
Shin, T., Kraemer, D., Pryor, J., et al. (2002). A cat cloned
by nuclear transplantation. Nature 415, 859.
Suemizu, H., Aiba, K., Yoshikawa, T., et al. (2003). Expression profiling of placentomegaly associated with
nuclear transplantation of mouse ES cells. Dev. Biol.
253, 3653.
Verhage, H.G., Beamer, N.B., and Brenner, R.M. (1976).
Plasma levels of estradiol and progesterone in the cat
during polyestrus, pregnancy and pseudopregnancy.
Biol. Reprod. 14, 579585.
Walker, S.C., Shin, T., Zaunbrecher, G.M., et al. (2002). A
highly efficient method for porcine cloning by nuclear
transfer using in vitromatured oocytes. Cloning Stem
Cells 4, 105112.
Walmsley, S.E., Buckrell, B.C., Buschbeck, C., et al. (2004).
Rate of abnormalities in lambs from in vitroproduced
embryos transferred on day 2 compared with day 6
postfertilization. Theriogenology 62, 195206.
Wells, D.N., Misica, P.M., Day, T.A., et al. (1997). Production of cloned lambs from an established embryonic
cell line: acomparison between in vivo and in vitromatured cytoplasts. Biol. Reprod. 57, 385393.
Wilmut, I., and Paterson, L. (2003). Somatic cell nuclear
transfer. Oncol. Res. 13, 303307.
Young, L.E., Fernandes, K., McEvoy, T.G., et al. (2001).
Epigenetic change in IGF2R is associated with fetal
overgrowth after sheep embryo culture. Nat. Genet. 27,
153154.

Address reprint requests to:

Dr. Martha C. Gmez
Audubon Center for Research of
Endangered Species
New Orleans, LA 70131
E-mail: mgomez@auduboninstitute.org

This article has been cited by:

1. Olga strup , Frantisek Strejcek , Ida Petrovicova , Andrea Lucas-Hahn , Martin Morovic , Erika Lemme , Bjorn Petersen , Nada
Laurincikova , Heiner Niemann , Jozef Laurincik , Poul Hyttel . 2011. Role of Ooplasm in Nuclear and Nucleolar Remodeling
of Intergeneric Somatic Cell Nuclear Transfer Embryos during the First Cell CycleRole of Ooplasm in Nuclear and Nucleolar
Remodeling of Intergeneric Somatic Cell Nuclear Transfer Embryos during the First Cell Cycle. Cellular Reprogramming
(Formerly "Cloning and Stem Cells") 13:2, 145-155. [Abstract] [Full Text] [PDF] [PDF Plus]
2. Nguyen Van THUAN, Satoshi KISHIGAMI, Teruhiko WAKAYAMA. 2010. How to Improve the Success Rate of Mouse
Cloning Technology. Journal of Reproduction and Development 56:1, 20-30. [CrossRef]
3. Raul E. Pia-Aguilar , Janet Lopez-Saucedo , Richard Sheffield , Lilia I. Ruiz-Galaz , Jose de J. Barroso-Padilla , Antonio
Gutirrez-Gutirrez . 2009. Revival of Extinct Species Using Nuclear Transfer: Hope for the Mammoth, True for the Pyrenean
Ibex, But Is It Time for Conservation Cloning?Revival of Extinct Species Using Nuclear Transfer: Hope for the Mammoth,
True for the Pyrenean Ibex, But Is It Time for Conservation Cloning?. Cloning and Stem Cells 11:3, 341-346. [Abstract]
[PDF] [PDF Plus]
4. Martha C. Gmez , Charles Earle Pope , Robert H. Kutner , David M. Ricks , Leslie A. Lyons , Mark T. Ruhe , Cherie Dumas
, Justine Lyons , Betsy L. Dresser , Jakob Reiser . 2009. Generation of Domestic Transgenic Cloned Kittens Using Lentivirus
VectorsGeneration of Domestic Transgenic Cloned Kittens Using Lentivirus Vectors. Cloning and Stem Cells 11:1, 167-176.
[Abstract] [PDF] [PDF Plus]
5. Goo JANG, SoGun HONG, JungTaek KANG, JungEun PARK, HyunJu OH, ChanKyu PARK, JiHong HA, DaeYong KIM,
MinKyu KIM, ByeongChun LEE. 2009. Conservation of the Sapsaree (Canis familiaris), a Korean Natural Monument, using
Somatic Cell Nuclear Transfer. Journal of Veterinary Medical Science 71:9, 1217-1220. [CrossRef]
6. Amy E. Iager , Neli P. Ragina , Pablo J. Ross , Zeki Beyhan , Kerrianne Cunniff , Ramon M. Rodriguez , Jose B. Cibelli . 2008.
Trichostatin A Improves Histone Acetylation in Bovine Somatic Cell Nuclear Transfer Early EmbryosTrichostatin A Improves
Histone Acetylation in Bovine Somatic Cell Nuclear Transfer Early Embryos. Cloning and Stem Cells 10:3, 371-380. [Abstract]
[PDF] [PDF Plus]
7. T Tsujioka, C Otzdorff, J Braun, S Hochi. 2008. Effect of Post-IVF Developmental Kinetics on In Vitro Survival of
Vitrified-warmed Domestic Cat Blastocysts. Reproduction in Domestic Animals 43:3, 323-327. [CrossRef]
8. Liesl Nel-Themaat , Martha C. Gmez , C. Earle Pope , Monica Lopez , Gemechu Wirtu , Alex Cole , Betsy L. Dresser ,
Leslie A. Lyons , Kenneth R. Bondioli , Robert A. Godke . 2008. Cloned Embryos from Semen. Part 1: In Vitro Proliferation of
Epithelial Cells on Embryonic Fibroblasts after Isolation from Semen by Gradient CentrifugationCloned Embryos from Semen.
Part 1: In Vitro Proliferation of Epithelial Cells on Embryonic Fibroblasts after Isolation from Semen by Gradient Centrifugation.
Cloning and Stem Cells 10:1, 143-160. [Abstract] [PDF] [PDF Plus]
9. Liesl Nel-Themaat , Martha C. Gmez , C. Earle Pope , Monica Lopez , Gemechu Wirtu , Jill A. Jenkins , Alex Cole , Betsy L.
Dresser , Kenneth R. Bondioli , Robert A. Godke . 2008. Cloned Embryos from Semen. Part 2: Intergeneric Nuclear Transfer of
Semen-Derived Eland (Taurotragus oryx) Epithelial Cells into Bovine OocytesCloned Embryos from Semen. Part 2: Intergeneric
Nuclear Transfer of Semen-Derived Eland (Taurotragus oryx) Epithelial Cells into Bovine Oocytes. Cloning and Stem Cells 10:1,
161-172. [Abstract] [PDF] [PDF Plus]
10. Ampika THONGPHAKDEE, Shuji KOBAYASHI, Kei IMAI, Yasushi INABA, Mariko TASAI, Takahiro TAGAMI, Keijiro
NIRASAWA, Takashi NAGAI, Norio SAITO, Mongkol TECHAKUMPHU, Kumiko TAKEDA. 2008. Interspecies Nuclear
Transfer Embryos Reconstructed from Cat Somatic Cells and Bovine Ooplasm. Journal of Reproduction and Development 54:2,
142-147. [CrossRef]
11. H Zhou, C Liu, W Wang. 2007. Heterospecific Nuclear-transferred Embryos Derived from Equine Fibroblast Cells and
Enucleated Bovine Oocytes. Reproduction in Domestic Animals 42:3, 243-247. [CrossRef]
12. Min Kyu Kim , Goo Jang , Hyun Ju Oh , Fibrianto Yuda , Hye Jin Kim , Woo Suk Hwang , Mohammad Shamim Hossein ,
Joung Joo Kim , Nam Shik Shin , Sung Keun Kang , Byeong Chun Lee . 2007. Endangered Wolves Cloned from Adult Somatic
CellsEndangered Wolves Cloned from Adult Somatic Cells. Cloning and Stem Cells 9:1, 130-137. [Abstract] [PDF] [PDF Plus]
13. Chang-Long NAN, Zi-Li LEI, Zhen-Jun ZHAO, Li-Hong SHI, Ying-Chun OUYANG, Xiang-Fen SONG, Qing-Yuan SUN,
Da-Yuan CHEN. 2007. Increased Th1/Th2 (IFN-/IL-4) Cytokine mRNA Ratio of Rat Embryos in the Pregnant Mouse
Uterus. Journal of Reproduction and Development 53:2, 219-228. [CrossRef]
14. Dr. Martha C. Gmez, , Charles Earle Pope , Mnica Lpez , C. Dumas , Angelica Giraldo , Betsy L. Dresser . 2006. Chromosomal
Aneuploidy in African Wildcat Somatic Cells and Cloned EmbryosChromosomal Aneuploidy in African Wildcat Somatic Cells
and Cloned Embryos. Cloning and Stem Cells 8:2, 69-78. [Abstract] [PDF] [PDF Plus]

15. A Thongphakdee, P Numchaisrika, S Omsongkram, K Chatdarong, S Kamolnorranath, S Dumnui, M Techakumphu. 2006. In

Vitro Development of Marbled Cat Embryos Derived from Interspecies Somatic Cell Nuclear Transfer. Reproduction in Domestic
Animals 41:3, 219-226. [CrossRef]