Theriogenology xxx (2015) 1–8

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Theriogenology
journal homepage: www.theriojournal.com

Ectopic liver and gallbladder in a cloned dog: Possible

nonheritable anomaly
Min Jung Kim a, Sang Chul Kang b, Jae Hwan Kim c, Hyun Ju Oh a,
Geon A Kim a, Young Kwang Jo a, Jin Choi a, Hyunil Kim b, Yeon Hea Lee c,
Ji Min Yoo c, Ki Dong Eom c, Byeong Chun Lee a, *
a
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Republic of
Korea
b
Department of Animal Clinical Evaluation, Optipharm, Inc., Cheongju, Chungcheongbuk-do, Republic of Korea
c
Department of Veterinary Radiology and Diagnostic Imaging, College of Veterinary Medicine, Konkuk University, Seoul, Republic of
Korea

a r t i c l e i n f o a b s t r a c t

Article history: Ectopic liver and gallbladder are rare anomalies usually not accompanied by any symptoms
Received 3 March 2015 and are found during surgical exploration or autopsy. We aimed to find a cause of this
Received in revised form 28 May 2015 anomaly using somatic cell nuclear transfer (SCNT) technology, which can produce
Accepted 28 May 2015
genetically identical organisms. A cloned beagle having ectopic organs was produced and
died on the day of birth. Major and ectopic organs were fixed and underwent histologic
Keywords:
analysis. SCNT was performed using cells derived from the dead puppy to produce
Dog
reclones. Normality of internal organs in the original donor dog and recloned dogs was
Ectopic liver
Ectopic gallbladder evaluated by computed tomography. While a liver without the gallbladder was located in
Etiology the abdominal cavity of the cloned dog, a well-defined, reddish brown mass with a small
Cloning sac was also positioned outside of the thoracic cavity. Histologically, they presented as
normal liver and gallbladder. Five reclones were produced, and computed tomography
results revealed that the original donor dog and reclones had normal liver and gallbladder
structure and location. This is the first report of both ectopic liver and gallbladder in an
organism and investigation on the etiology of these abnormalities. Normal organ structure
and position in the original donor dog and reclones suggests that the ectopic liver and
gallbladder is a possible nonheritable anomaly.
Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction
The term “choristoma” originally comes from the
German word “to separate” and was introduced by Eugen
Albrecht in 1904 to refer to a mass of normal tissue located
at a site away from its usual location [1]. Choristoma
occurring with hepatic or gallbladder tissue is commonly
called ectopic or heterotopic liver or gallbladder. Ectopic
liver and gallbladder have been recorded since the 19th
* Corresponding author. Tel.: þ822 880 1269; fax: þ822 873 1269.
E-mail address: bclee@snu.ac.kr (B.C. Lee).
century [2], but only less than 80 cases of ectopic liver were
documented in a recent literature review [3] with lower
than 0.5% [4] and 0.4% [5] incidences. Ectopic liver has been
found in various sites including the gallbladder [3,6,7],
spleen [8], pancreas [9], stomach [10], heart [11], lung [12]
and suprahepatic inferior vena cava [13,14], and ectopic
gallbladder has been described in the intrahepatic [15] and
suprahepatic region [16], and left hepatic lobe [17,18]. Both
anomalies are usually asymptomatic, but rare symptoms
such as intra-abdominal bleeding due to ectopic liver [10]
or epigastric pain caused by ectopic gallbladder [17] have
been reported. Lack of symptoms usually results in
discovery of these anomalies only during peritoneoscopy,

0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2015.05.039
2 M.J. Kim et al. / Theriogenology xxx (2015) 1–8
laparotomy, or autopsy. Also, almost all the documented
cases of these anomalies have been described in human
beings, not in animals [1–6,8–12,15–21]. In dogs, ectopic
hepatocytes were firstly reported in 2005, in which a
bearded collie had a firm and nonpainful mass in the left
midabdominal region [22]. Consequently, scarcity of the
anomaly, lack of symptoms, and absence of animal models
have made it hard to define a cause of ectopic liver and
gallbladder.
Somatic cell nuclear transfer (SCNT) is a process for
producing genetically identical organisms asexually [23]
which includes nuclear removal from a donor oocyte,
donor cell injection into the empty perivitelline space,
fusion between the cytoplast–cell couplet, and activation
of the reconstructed embryo. Since the first cloned animal,
Dolly the sheep, was produced using SCNT in 1997 [24],
more than 16 mammalian species including mice [25], rats
[26], cattle [27], pigs [28], cats [29], and dogs [30] have
been successfully produced. Among these, dogs have ad-
vantages as animal models for human diseases because of
the similarities in their size, longevity, and physiology to
humans. Of the nearly 648 known hereditary diseases
described in dogs, more than half (352) can be potential
models for human diseases (http://omia.angis.org.au,
January 2014). A human disease model dog can be gener-
ated by replacing a donor oocyte’s nucleus with a cell
derived from a dog naturally having a heritable disease or
with a cell containing a genetically modified transgene
[31,32]. For example, a cloned puppy derived from a donor
dog having hip dysplasia, which is an inherited disease
characterized by hip subluxation and laxity [33,34], also
showed signs of hip dysplasia. Because of the identical
genetic traits between donor and cloned dogs, SCNT can be
used as a tool for studying potential causes of unidentified
disease to determine whether it is caused by a genetic
modification or not.
In the present study, we report for the first time an
occurrence of both ectopic liver and gallbladder in a cloned
dog and aimed to investigate their etiology by recloning
using cells derived from the clone.
2. Materials and methods
2.1. Animal use
Animal experiments were done following a standard
procedure established by the Committee for Accreditation
of Laboratory Animal Care and the Guideline for the Care
and Use of Laboratory Animals of Seoul National University
(approval number is SNU-121130-1).
2.2. Production of a cloned dog
Ear skin tissue from a 10-year-old male beagle (Fig. 1A)
was collected and transferred to the laboratory aseptically.
The tissue was minced and cultured with Dulbecco’s
modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 10% (v:v) fetal bovine serum (Invi-
trogen). Donor cells were cultured to confluence and
retrieved as single cells by trypsinization just after recovery
of in vivo–matured oocytes.
Recovery of in vivo–matured oocytes, SCNT, and embryo
transfer were done on the basis of a previous report
[30,35]. In brief, surgical oviduct flushing was performed
72 hours after serum progesterone reached 5 to 10 mg/mL
[36], and cumulus cells of matured oocytes were removed
by repeated pipetting in 0.1% (wt/vol) hyaluronidase in
TCM-199. Denuded oocytes underwent enucleation in
TCM-199 containing 5 mg/mL of cytochalasin B and 5 mg/mL
of bisbenzimide. After injection of a donor cell into the
enucleated oocyte, the oocyte–cell couplet was fused with
electrical stimulation (2 pulses of 72 V for 15 ms) and
activated with 10-mM calcium ionophore and 1.9-mM 6-
dimethylaminopurine. Then, the cloned embryos were
surgically transferred into one oviduct of a naturally syn-
chronized recipient dog. Pregnancy diagnosis was per-
formed at least 28 days after the embryo transfer by
ultrasonography, and serum progesterone concentration,
rectal temperature, and fetal heartbeat were monitored for
safe delivery [37].
2.3. Recloning and microsatellite (MS) analysis
Skin tissue was collected aseptically from a neonatal dog
having ectopic organs on the day of birth, and fibroblasts
were acquired by the same methods used for the original
donor dog. Recloning also followed the same methods
described for production of a cloned dog.
Parentage analysis was performed on the original donor
dog, the cloned dog, and recloned dogs to confirm their
genetic identity. Eight MS markers including PEZ1, PEZ3,
PEZ5, PEZ8, PEZ2, FHC2010, FHC2054, and FHC2079 [38]
were chosen for analysis. The isolated genomic DNA sam-
ples were dissolved in 50-mL TE (10 mM Tris, 1 mM EDTA,
pH 8.0), and length variations were assayed by polymerase
chain reaction amplification with fluorescently labeled
(FAM, HEX, and NED) locus-specific primers and PAGE on
an automated DNA sequencer (ABI 373; Applied Bio-
systems, Foster City, CA, USA). Proprietary software (Gen-
eScan and Genotyper; Applied Biosystems) was used to
estimate the polymerase chain reaction product size in
nucleotides.
2.4. Histologic analysis
Organ samples including the heart, lung, spleen, kidney,
livers in normal location (mother liver) and ectopic site
(ectopic liver), and gallbladder of the cloned dog were fixed
in 4% paraformaldehyde until analyzed. After slicing the
samples with similar thickness for further processing, the
sliced pieces were dehydrated. Subsequently, the pieces
were embedded in paraffin wax and cut into 5-mm-thick
sections using a microtome. The sections were stained with
hematoxylin and eosin and examined under light micro-
scopy to assess histologic normality of the major and
ectopic organs.
2.5. Computed tomography
The computed tomography (CT) was performed on the
original donor dog (aged 11 years) and recloned dogs
(aged 1 year) to compare normality of their internal
 M.J. Kim et al. / Theriogenology xxx (2015) 1–8 3

Fig. 1. Photographs of original cell donor dog at 10 years of age (A) and recloned dogs (B and C). Re2 had congenital hind bow-legged malformation (B), but others
(Re1, 3, 4, and 5) exhibited normal appearance.

organs, especially the liver and gallbladder. After a com-
plete blood cell count and serum chemistry analysis, the
CT was performed with a helical CT scanner (GE CT/e;
General Electronic Medical Systems, WI, USA) under gen-
eral anesthesia. After unenhanced images of the whole
body were obtained, triple-phase dynamic CT of the liver
was done. Using a power injector (CT 9000 Digital Injec-
tion System; Liebel-Flarsheim, Cincinnati, OH, USA),
contrast agent iohexol (Omnipaque 300, Daiichi Seiyaku,
Tokyo, Japan) was administered at rates of 2 mL/s into the

Fig. 2. Autopsy of the cloned dog that died due to cannibalism by a nanny dog on the day of birth. (A) Ectopic liver and gallbladder with smooth margin
positioned between ribs and skin. (B) Bite wounds (a–c) from its nanny dog.
4 M.J. Kim et al. / Theriogenology xxx (2015) 1–8

Fig. 3. Histologic appearance of mother liver (A), ectopic liver (B), ectopic gallbladder (C), and kidney (D) of the cloned dog. Although normal histologic ar-
chitectures were seen in the mother liver, ectopic liver, and ectopic gallbladder, chronic pathologic changes were found in the kidney including multifocal tubular
degeneration and cystic dilatation (C) in the cortex and intralesional fibroplasia in the interstitium.

left cephalic vein. In all dogs, aortic enhancement was
identified using the SmartPrep (GE Medical Systems)
technique. Arterial-phase CT was initiated 2 seconds after
identification of onset of the aortic enhancement. Portal
venous-phase and equilibrium-phase scans were initiated
20 and 100 seconds after the start of the arterial-phase
scan, respectively. Hepatic vessels and patterns of
contrast enhancement were evaluated with triple-phase
helical CT examination.
3. Results
3.1. Gross pathologic examination of ectopic organs found in a
cloned dog
A cloned dog died on the day of birth because of
cannibalism by its nanny dog. During the routine autopsy,
ectopic organs assumed to be liver and gallbladder were
found between the left-side ribs and the skin (Fig. 2A).
Although there was a properly located liver (mother liver)
with six lobes, no gallbladder was present in the abdominal
cavity. Interestingly, there was a well-defined, large solid
mass (6  4  0.5 cm) with a smooth surface and reddish
brown color similar to the normal liver (ectopic liver)
located subcutaneously outside of the thoracic cavity. It
was composed of three lobes, and a small sac having similar
morphology and color to a gallbladder was connected to
one of the lobes. Connections between the ectopic liver and
the mother liver could not be found, and no other abnor-
malities were found. Two perforations were observed in
the abdominal wall near the right flank and one more near
the 10th intercostal muscle (Fig. 2B), which might be bite
wounds. The largest perforation was on the left flank and
was 1 cm wide.
3.2. Ectopic liver and gallbladder determined by histologic
examination
The ectopic reddish brown mass had hepatic lobules
with a central vein and portal canals like the mother liver in
the abdominal cavity. Parenchyma of both organs con-
tained swollen hepatocytes with clear cytoplasm and bile
ducts (Fig. 3A, B). There was no histologically remarkable
difference between these two organs and no evidence of
malignancy. Cross sections of the small sac could be divided
into three layers: mucosa, muscularis externa, and adven-
titia (Fig. 3C), the same as found in a normal gallbladder.
The lumen of the sac was lined with a high columnar
epithelium with microvilli and lamina propria seen un-
derneath the same epithelium. There was no pathologic
change in this organ, either. However, chronic pathologic
changes including multifocal tubular degeneration and
cystic dilatation in the cortex and intralesional fibroplasia
in the interstitium, which are evidence of nephrosis, were
 M.J. Kim et al. / Theriogenology xxx (2015) 1–8 5

Table 1 homogeneously after contrast medium administration. On

Production of recloned dogs using donor cells derived from a cloned dog
having an ectopic liver and gallbladder.
Recipient Recloned Delivery Birth Appearance at birth
ID dogs ID method weight
(g)
1 Re1 Natural 510 Normal
Re2 Natural 250 Bow-legged malformation of
hind legs (dead at Day 10)
2 Re3 Natural 460 Normal
Re4 C-sec 400 Normal
Re5 C-sec 410 Normal
Abbreviation: C-sec, cesarean section.
found in the kidney (Fig. 3D). There were no pathologic
changes in the heart, lung, or spleen (data not shown).
3.3. Production of recloned dogs
Using cells derived from the dead cloned dog as nuclear
donors, a total of 198 oocyte–cell couplets were produced,
151 embryos were fused (76.3% of fusion rate), and 132
embryos were transferred to nine recipients. Two recloned
pups were naturally delivered from a recipient, whereas
three were delivered both naturally and by cesarean section
from another recipient (Table 1). Among them, one was
born weak with a birth weight of 250 g (Fig. 1B), whereas
others had an average weight of 370 g, ranging from 400 to
510 g (Fig. 1C). The weak puppy was also hind bow legged
which made it hard to competitive with littermates. It died
on Day 10 after birth, and no remarkable abnormality
including liver and gallbladder position was found during
autopsy. The other four recloned dogs are still alive and
healthy.
3.4. MS and CT results
The four recloned dogs, the cloned dog, and the original
donor dog had identical MS patterns for all loci (Table 2).
Serum chemistry analysis including aspartate aminotrans-
ferase, alanine aminotransferase, alkaline phosphatase,
gamma-glutamyl transferase, bilirubin, blood urea nitro-
gen, and creatinine and blood cell count results were
within normal reference ranges (data not shown). The CT
images revealed that all the recloned dogs and the original
donor dog had normal liver and gallbladder structure and
position (Fig. 4). The CT scans did not show any abnormal
findings related to hepatobiliary systems. All liver lobes
were isoattenuated on precontrast and enhanced
triple-phase CT, all dogs had normal anatomic structure
including the hepatic artery, and portal and hepatic veins.
In addition, the gallbladder was located at the normal po-
sition between the right medial hepatic lobe and quadrate
hepatic lobe in all dogs.
4. Discussion
Ectopic organs are rare congenital abnormalities.
Several organs including the liver [1–6,8–12,15–19], gall-
bladder [5,17,19], pancreas [39], kidney [40], testis [41], and
ovary [42] located in ectopic positions have been reported
in humans. Because the aberrant tissue is often not
accompanied by any clinical relevance [3,19,39], it has a low
incidence rate and it is difficult to analyze the reason for its
occurrence. Thus, in the present study, we aimed to report
an incidental finding of ectopic liver and gallbladder in a
cloned dog and to give a clue to its occurrence by recloning
the cloned dog and analyzing the cell donor and recloned
dogs.
Collan et al. [43] classified abnormal human liver tissue
into four main types: type I is a large accessory liver lobe
attached to the mother liver by a stalk, type 2 is a small
accessory liver lobe attached to the mother liver, type 3 is
an ectopic liver without any connection to the mother liver
and is usually attached to the gallbladder or intra-
abdominal ligaments, and type 4 is a microscopic ectopic
liver in the wall of the gallbladder. In our study, although
there were several bite wounds in the cloned dog, the
ectopic liver found in this animal belongs to type 3. It is
certain that the liver and gallbladder were located in an
aberrant position because of developmental problems and
not because of the injuries because the bite wounds were
too small to pass the large liver and most of the surface of
the ectopic and mother livers was smooth (Fig. 2). Although
ectopic liver usually has histologically normal hepatic ar-
chitecture, it can be subject to histopathologic changes
resembling carcinogenesis probably because of a compro-
mised vascular supply or impaired biliary drainage [9]. It
seems that ectopic liver could be larger than
5.0  5.0  5.0 cm when it contains hepatocellular carci-
noma [8,9], but ectopic liver of the cloned dog was rela-
tively large even without histopathologic changes (Fig. 3B)
in either the ectopic or mother liver. This could be the result
of sufficient space for the ectopic liver to grow compared to
constraints within internal cavities containing various
organs.

Table 2
Microsatellite (MS) analysis of the original cell donor, the cloned dog having ectopic liver and gallbladder, and four recloned dogs (Re1, 3, 4, and 5).

MS marker PEZ1 PEZ3 PEZ5 PEZ8 PEZ20 FHC2010 FHC2054 FHC2079

Control 119/123 115/121 103/103 230/238 172/180 223/233 151/171 274/274
Cell donor 115/119 124/136 103/107 238/242 176/180 229/237 155/167 270/270
Clone 115/119 124/136 103/107 238/242 176/180 229/237 155/167 270/270
Re1 115/119 124/136 103/107 238/242 176/180 229/237 155/167 270/270
Re3 115/119 124/136 103/107 238/242 176/180 229/237 155/167 270/270
Re4 115/119 124/136 103/107 238/242 176/180 229/237 155/167 270/270
Re5 115/119 124/136 103/107 238/242 176/180 229/237 155/167 270/270
6 M.J. Kim et al. / Theriogenology xxx (2015) 1–8
Fig. 4. Computed tomography scans showing normal liver position (upper row) and structure (lower row) of the original cell donor and recloned dogs (Re1, 3, 4,
and 5). No abnormal findings related to hepatobiliary systems including the liver, gallbladder, and vessels were apparent. GB, gallbladder; LLL, left lateral lobe;
LML, left medial lobe; RLL, right lateral lobe; RML, right medial lobe; QL, quadrate lobe.
Important congenital abnormalities of a gallbladder are
related to number (agenesis, bilobed or multiseptate gall-
bladder), shape (Phrygian cap, diverticulum, or hypoplasia),
or position (ectopic gallbladder) [5,44]. The four most
common ectopic locations are under the left liver lobe,
inside the liver, transverse, and retroplaced [18,19,45–47].
However, there is an exceptional case which did not belong
to this classification. Alkatout et al. [5] reported a small,
stalked, pear-shaped gallbladder on the right side of the
umbilical cord in a newborn boy. Similar to this, the ectopic
gallbladder of the cloned dog was placed external to the
thoracic cavity with a connection to the ectopic liver. A
review article described that left-sided gallbladder is prone
to diseases including cholelithiasis, polyps, and empyema
(seven among 110 cases) [18]. Ectopic gallbladder of the
cloned dog showed no pathologic changes such as a gall
stone or inflammation (Fig. 3C). Normal histologic
appearance in the mother liver, ectopic liver, and ectopic
gallbladder (Fig. 3A, B, C) might imply that the duration of
liver and gallbladder development during embryogenesis is
not enough to cause any pathologic changes in these
organs. Although chronic nephrosis was observed in the
kidney of the cloned dog, the relationship between ectopic
organs (liver and gallbladder) and chronic nephrosis is not
clear.
During embryogenesis, rudimentary buds of the endo-
dermal caudal foregut (primitive foregut) grows upward
and laterally into the adjacent mesenchyme (the septum
transversum) [48]. The hepatic diverticulum differentiates
into the pars hepatica (cranial part; future liver paren-
chyma) and the pars cystica (caudal part; future gallbladder
and cystic duct) [48]. Both congenital displacements of the
liver and gallbladder in the cloned dog might have resulted
from aberrant differentiation of the rudimentary budding
of the endodermal caudal foregut in the early stage of
embryogenesis or abnormal movement of both pars
hepatica and pars cystica. Up to now, there have been
reports of either ectopic liver or gallbladder, not both.
Although a reasonable hypothesis could be derived from
the embryogenesis information, there is no scientifically
based explanation of how these variations arise. Therefore,
we recloned the cloned dog using SCNT and examined the
cell donor and recloned dogs by CT.
Theoretically, clones and their nuclear donor animals
have the same genome, but cloned mammals occasionally
show developmental anomalies. The “large offspring syn-
drome” characterized by fetal and placental overgrowth or
other anomalies include respiratory distress, major car-
diovascular abnormalities, and enlargement of organs are
commonly seen in cloned ruminants [49]. Compared to
observations in ruminants, there have been few studies
related to congenital abnormalities in cloned dogs; pla-
centomegaly [50], malformations [32], cleft palate and
preputial abnormalities [51], and defects in the anterior
abdominal wall, increased heart and liver sizes, muscle
mass, and macroglossia have all been reported [52]. Aber-
rant epigenetic reprogramming in DNA methylation, his-
tone acetylation, and X-chromosome inactivation have
been proposed as the cause of developmental anomalies
seen in cloned animals [53]. Ectopic liver and gallbladder in
our case is a new kind of abnormality found in a cloned dog
and is an extremely rare congenital anomaly in naturally
produced dogs. We hypothesized that if these develop-
mental problems were due to aberrant epigenetic reprog-
ramming, the cell donor and recloned dogs would have
normally positioned livers and gallbladders. In accordance
with our hypothesis, all of them had normal anatomical
liver structure including the hepatic artery and the portal
and hepatic veins as well as the gallbladder located in its
normal position (Fig. 4) and also had normal serum
chemistry values for aspartate aminotransferase, alanine
aminotransferase, alkaline phosphatase, gamma-glutamyl
transferase, bilirubin, blood urea nitrogen, and creatinine.
Interestingly, a recloned puppy showed a different kind of
 M.J. Kim et al. / Theriogenology xxx (2015) 1–8
anomaly, bow-legged hind legs, which is often seen in
natural breeding or artificial insemination [54] but has
never been reported in a cloned dog. Also, congenital
malformations in one cloned pup out of five seem more
frequent than in previous studies (one out of 33 non-
transgenic clones in a review article [55]). Therefore, it
could be suggested that aberrant reprogramming in a
cloned dog can be corrected via SCNT but could cause other
types of reprogramming errors.
In conclusion, this is the first study to report an occur-
rence of both ectopic liver and gallbladder together and
malposition of these ectopic organs in an extrathoracic
location, between ribs and skin. Successful reprogramming
in the recloned dogs could reflect that ectopic organs found
in our cloned dog were possibly a nonheritable anomaly.
Our study provides a clue that ectopic livers and gallblad-
ders found in human could be the result of an aberrant
epigenetic modification during embryogenesis, but further
studies about causes leading to those congenital anomalies
are needed because congenital anomalies are probably
caused by multifactorial etiology [56–58].
Acknowledgments
This study was supported by Rural Development
Administration (#PJ010928032015), Korea Institute of
Planning and Evaluation for Technology in Food, Agricul-
ture, Forestry and Fisheries (#311062-04-3SB010), National
Research Foundation of Korea (#2014R1A1A2059928),
Research Institute for Veterinary Science, Natural Balance
Korea, and the BK21 plus program. The authors thank Dr
Barry D. Bavister for his valuable editing of the article.
Competing Interests
None of the authors of this article has a financial or
personal relationship with other people or organizations
that could inappropriately influence or bias the content of
the article.
References
[1] Albrecht E. Uber Hamartome. Verh Dtsch Ges Pathol 1904;7:153–7.
[2] Cullen TS. Accessory lobes of the liver: an accessory hepatic lobe
springing from the surface of the gallbladder. Arch Surg 1925;11:
718–64.
[3] Catani M, De Milito R, Romagnoli F, Mingazzini P, Silvestri V, Usai V,
et al. Ectopic liver nodules: a rare finding during cholecystectomy. G
Chir 2011;32:255–8.
[4] Watanabe M, Matsura T, Takatori Y, Ueki K, Kobatake T, Hidaka M,
et al. Five cases of ectopic liver and a case of accessory lobe of the
liver. Endoscopy 1989;21:39–42.
[5] Alkatout I, Henopp T, Moritz JD, Nikischin W, Klöppel G, Engler S.
Extraabdominal malposition of the gallbladder. J Pediatr Surg 2008;
43:e41–4.
[6] Martinez CA, de Resende Júnior HC, Rodrigues MR, Sato DT,
Brunialti CV, Palma RT. Gallbladder-associated ectopic liver: a rare
finding during a laparoscopic cholecystectomy. Int J Surg Case Rep
2013;4:312–5.
[7] Karaman K, Teke Z, Ercan M, Keklik TT, Bostanci EB, Akoglu M.
Ectopic liver (choristoma) attached to the gallbladder wall. ANZ J
Surg 2012;82:948.
[8] Kim KA, Park CM, Kim CH, Choi SY, Park SW, Hong SJ, et al. Hepa-
tocellular carcinoma in an ectopic liver: CT findings. Eur Radiol
2003;13:L45–7.
7
[9] Kubota K, Kita J, Rokkaku K, Iwasaki Y, Sawada T, Imura J, et al.
Ectopic hepatocellular carcinoma arising from pancreas: a
case report and review of the literature. World J Gastroenterol 2007;
13:4270.
[10] Arakawa M, Kimura Y, Sakata K, Kubo Y, Fukushima T, Okuda K.
Propensity of ectopic liver to hepatocarcinogenesis: case reports
and a review of the literature. Hepatology 1999;29:57–61.
[11] Brustmann H. Heterotopic liver in the right cardiac auricle. Ann
Diagn Pathol 2002;6:248–9.
[12] Robertson DJ, Harmon CM, Goldberg S. Right congenital diaphrag-
matic hernia associated with fusion of the liver and the lung. J
Pediatr Surg 2006;41:e9–10.
[13] Morris MW, Helling TS, Creswell LL, Jordan B, Mitchell ME. Ectopic
liver masquerading as a floating intracaval mass. J Vasc Surg 2012;
55:1759–61.
[14] Rafiei P, Sebastian S, Patel RB, Roda MS. Ectopic intracaval liver. Clin
Imaging 2012;36:869–72.
[15] Schulz RC, Shields JB, Fletcher JW, Donati RM. Liver scanning and
the intrahepatic gallbladder: case report. J Nucl Med 1975;16:
1029–30.
[16] Youngwirth L, Peters J, Perry M. The suprahepatic gallbladder. An
unusual anatomical variant. Radiology 1983;149:57–8.
[17] Chung C, Leung K, Lau W, Li AK. Ectopic gallbladder revisited, lap-
aroscopically: a case report. Can J Surg 1997;40:464.
[18] Dhulkotia A, Kumar S, Kabra V, Shukla H. Aberrant
gallbladder situated beneath the left lobe of liver. HPB (Oxford)
2002;4:39–42.
[19] Rafailidis V, Varelas S, Kotsidis N, Rafailidis D. Two congenital
anomalies in one: an ectopic gallbladder with phrygian cap defor-
mity. Case Rep Radiol 2014;2014:246476.
[20] van Kamp M-J, Bouman DE, Steenvoorde P, Klaase JM. A Phrygian
cap. Case Rep Gastroenterol 2013;7:347–51.
[21] de Csepel J, Pomp A. Soft-tissue images.“Phrygian cap” gallbladder.
Can J Surg 2003;46:50.
[22] Burton I, Limpus K, Thompson K, Owen M, Worth A. Ectopic hepa-
tocellular carcinoma in a dog. N Z Vet J 2005;53:465–7.
[23] Di Berardino MA. Animal cloningdthe route to new genomics in
agriculture and medicine. Differentiation 2001;68:67–83.
[24] Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable
offspring derived from fetal and adult mammalian cells. Nature
1997;385:810–3.
[25] Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R. Full-
term development of mice from enucleated oocytes injected with
cumulus cell nuclei. Nature 1998;394:369–74.
[26] Zhou Q, Renard J-P, Le Friec G, Brochard V, Beaujean N, Cherifi Y,
et al. Generation of fertile cloned rats by regulating oocyte activa-
tion. Science 2003;302:1179.
[27] Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, et al.
Cloned transgenic calves produced from nonquiescent fetal fibro-
blasts. Science 1998;280:1256–8.
[28] Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, et al.
Cloned pigs produced by nuclear transfer from adult somatic cells.
Nature 2000;407:86–90.
[29] Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, et al. Cell biology:
a cat cloned by nuclear transplantation. Nature 2002;415:859.
[30] Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, et al. Dogs cloned
from adult somatic cells. Nature 2005;436:641.
[31] Hong SG, Kim MK, Jang G, Oh HJ, Park JE, Kang JT, et al. Generation
of red fluorescent protein transgenic dogs. Genesis 2009;47:314–22.
[32] Kim MJ, Oh HJ, Park JE, Kim GA, Hong SG, Jang G, et al. Generation of
transgenic dogs that conditionally express green fluorescent pro-
tein. Genesis 2011;49:472–8.
[33] Wilson BJ, Nicholas FW, James JW, Wade CM, Raadsma HW,
Thomson PC. Genetic correlations among canine hip dysplasia
radiographic traits in a cohort of Australian German Shepherd dogs,
and implications for the design of a more effective genetic control
program. PLoS One 2013;8:e78929.
[34] Lewis TW, Blott SC, Woolliams JA. Comparative analyses of genetic
trends and prospects for selection against hip and elbow dysplasia
in 15 UK dog breeds. BMC Genet 2013;14:16.
[35] Kim MJ, Oh HJ, Park JE, Hong SG, Kang JT, Koo OJ, et al. Influence of
oocyte donor and embryo recipient conditions on cloning efficiency
in dogs. Theriogenology 2010;74:473–8.
[36] Kim M-J, Oh H-J, Kim G-A, Jo Y-K, Choi J, Lee B-C. Application of
chemiluminescence enzyme immunoassay method to collect in vivo
matured oocyte in dog cloning. J Vet Clin 2014;31:267–71.
[37] Kim MJ, Oh HJ, Park JE, Kim GA, Park EJ, Jo YK, et al. Duration of
gestation in pregnant dogs carrying cloned fetuses. Theriogenology
2013;79:257–60.
8 M.J. Kim et al. / Theriogenology xxx (2015) 1–8
[38] Oh HJ, Park JE, Kim MJ, Hong SG, Ra JC, Jo JY, et al. Recloned dogs
derived from adipose stem cells of a transgenic cloned beagle.
Theriogenology 2011;75:1221–31.
[39] Thoeni RF, Gedgaudas RK. Ectopic pancreas: usual and unusual
features. Gastrointest Radiol 1980;5:37–42.
[40] Collura G, De Dominicis M, Patricolo M, Caione P. Hydronephrosis
due to malrotation in a pelvic ectopic kidney with vascular anom-
alies. Urol Int 2003;72:349–51.
[41] Oludiran O, Sakpa C. Crossed ectopic testis: a case report and review
of the literature. Pediatr Surg Int 2005;21:672–3.
[42] Watkins B, Kothari S. True ectopic ovary: a case and review. Arch
Gynecol Obstet 2004;269:145–6.
[43] Collan Y, Hakkiluoto A, Hästbacka J. Ectopic liver. Ann Chir Gynaecol
1977;67:27–9.
[44] Kachare MB, Khandelwal A, Kulkarni SB, Saboo SS. Ectopic thyroid in
duplicated gall bladder: a rare entity. Case report. Med Ultrason
2013;15:73–5.
[45] Kawai R, Miyata K, Yuasa N, Takeuchi E, Goto Y, Miyake H, et al. True
left-sided gallbladder with a portal anomaly: report of a case. Surg
Today 2012;42:1130–4.
[46] Shimizu T, Hayashi M, Inoue Y, Komeda K, Asakuma M, Hirokawa F,
et al. Living-donor liver transplantation from donor with a left-sided
gallbladder with portal vein anomaly. Transplantation 2012;94:
e60–1.
[47] Strong RW, Fawcett J, Hatzifotis M, Hodgkinson P, Lynch S,
O’Rourke T, et al. Surgical implications of a left-sided gallbladder.
Am J Surg 2013;206:59–63.
[48] Moore KL, Persaud TVN, Torchia MG. The developing human. Ninth
edition. Philadelphia: Elsevier Health Sciences; 2011.
[49] Wilmut I, Beaujean N, De Sousa P, Dinnyes A, King T, Paterson L,
et al. Somatic cell nuclear transfer. Nature 2002;419:583–7.
[50] Oh HJ, Hong SG, Park JE, Kang JT, Kim MJ, Kim MK, et al. Improved
efficiency of canine nucleus transfer using roscovitine-treated
canine fibroblasts. Theriogenology 2009;72:461–70.
[51] Kim MJ, Oh HJ, Kim GA, Jo YK, Choi J, Kim HJ, et al. Reduced birth
weight, cleft palate and preputial abnormalities in a cloned dog.
Acta Vet Scand 2014;56:18.
[52] Hong IH, Jeong YW, Shin T, Hyun SH, Park JK, Ki MR, et al.
Morphological abnormalities, impaired fetal development and
decrease in myostatin expression following somatic cell nuclear
transfer in dogs. Mol Reprod Dev 2011;78:337–46.
[53] Niemann H, Tian XC, King WA, Lee RS. Epigenetic reprogramming in
embryonic and foetal development upon somatic cell nuclear
transfer cloning. Reproduction 2008;135:151–63.
[54] Kim M-J, Park S-J, Kim G-A, Park E-J, Moon J-H, Choi J-Y, et al. Failure
of reproduction management in an inbreeding English Bulldog. J Vet
Clin 2013;30:384–6.
[55] Kim M, Oh H, Kim G, Park J, Park E, Jang G, et al. Lessons learned
from cloning dogs. Reprod Domest Anim 2012;47:115–9.
[56] Pinborg A, Henningsen A-KA, Malchau SS, Loft A. Congenital
anomalies after assisted reproductive technology. Fertil Steril 2013;
99:327–32.
[57] Sheridan E, Wright J, Small N, Corry PC, Oddie S, Whibley C, et al.
Risk factors for congenital anomaly in a multiethnic birth cohort: an
analysis of the Born in Bradford study. Lancet 2013;382:1350–9.
[58] Rankin J, Nieuwenhuijsen M. Ambient air pollution and risk of
congenital anomalies: a systematic review and meta-analysis. En-
viron Health Perspect 2011;119:599.