Goo Jang

Dogs cloned from adult somatic cells

Several mammals — including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats — have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

Effects of melatonin on in vitro maturation of porcine oocyte and expression of melatonin receptor RNA in cumulus and granulosa cells.

Abstract:  Melatonin is a multifunctional molecule that mediates several circadian and seasonal processes in animal reproduction. Melatonin and its metabolites are antioxidants and free radical scavengers. We investigated the effects of melatonin on porcine oocyte maturation and embryo development. We then investigated the local expression of the melatonin receptor 1 (MT1) gene in cumulus cells, granulosa cells, and the oocytes with the reverse transcription‐polymerase chain reaction (RT‐PCR) method. We further evaluated the antioxidant effects [reactive oxygen species (ROS) levels in cumulus‐oocytes complexes] of melatonin supplementation during in vitro maturation (IVM). Compared with control, melatonin supplementation (10 ng/mL) during IVM resulted in a greater proportion of oocytes extruding the polar body (75.6% versus 84.6%). Significantly greater proportion of parthenogenetically activated oocytes developed to blastocysts when the in vitro medium was supplemented with melatonin; however, cleavage frequency and blastocyst cell number were not affected by the treatment. RT‐PCR analysis revealed the expression of MT1 gene in cumulus and granulosa cells but not in oocytes. Melatonin‐treated oocytes had significantly lower levels of ROS than did control (untreated) oocytes. We conclude that exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine IVM. Some of the observed effects may be mediated by receptor binding and while others may have been receptor independent, e.g., direct free radical scavenging.

Transglutaminase 2 suppresses apoptosis by modulating caspase 3 and NF-κB activity in hypoxic tumor cells

The expression of hypoxia-inducible factor-1 (HIF-1) correlates with poor clinical outcomes and confers resistance to the apoptosis of the tumor cells that are exposed to hypoxia. Presently, the mechanism underlying this phenomenon is poorly understood. In this study we provide evidence that transglutaminase 2 (TG2), an enzyme that catalyses protein crosslinking reactions, is a transcriptional target of HIF-1 to enhance the survival of hypoxic cells. We found that hypoxia induces TG2 expression through an HIF-1 dependent pathway and concurrently activates intracellular TG2. The hypoxic cells overexpressing TG2 showed resistance to apoptosis. Conversely, the hypoxic cells treated with either TG2 inhibitor or small interfering RNA (siRNA) became sensitive to apoptosis. Activation of TG2 in response to hypoxic stress inhibited caspase-3 activity by forming crosslinked multimer, resulting in insoluble aggregates. TG2 also activates nuclear factor (NF)-κB pathway after hypoxic stress, and thereby induces the expression of cellular inhibitor of apoptosis 2. The anti-apoptotic role of TG2 was further confirmed in vivo using xenografts in athymic mice. Our results indicate that TG2 is an anti-apoptotic mediator of HIF-1 through modulating both apoptosis and survival pathways and may confer a selective growth advantage to tumor cells. These findings suggest that the inhibition of TG2 may offer a novel strategy for anticancer therapy.

Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem

The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.

Generation of red fluorescent protein transgenic dogs.

Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene‐expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP‐fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP. genesis 47:314–322, 2009.

Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are attractive candidates for clinical repair or regeneration of damaged tissues. Oct4 and Sox2, which are essential transcription factors for pluripotency and self-renewal, are naturally expressed in MSCs at low levels in early passages, and their levels gradually decrease as the passage number increases. Therefore, to improve MSC proliferation and stemness, we introduced human Oct4 and Sox2 for conferring higher expansion and differentiation capabilities. The Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 were successfully transfected into ATMSCs, and we confirmed maintenance of MSC surface markers without alterations in both red fluorescent protein (RFP) (control) and Oct4/Sox2-ATMSCs. Enhanced proliferative activity of Oct4/Sox2-ATMSCs was shown by WST-1 assay, and this result was further confirmed by cell counting using trypan blue exclusion for a long period. In addition, FACs cell cycle analysis showed that there was a reduction in the fraction of Oct4/Sox2-ATMSCs in G1 with a concomitant increase in the fraction of cells in S, compared with RFP-ATMSCs. Increased levels of cyclin D1 were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Furthermore, Oct4/Sox2-overexpressing ATMSCs showed higher differentiation abilities for adipocytes or osteoblasts than controls. The markers of adipogenic or osteogenic differentiation were also upregulated by Oct4/Sox2 overexpression. The improvement in cell proliferation and differentiation using Oct4/Sox2 expression in ATMSCs may be a useful method for expanding the population and increasing the stemness of ATMSCs.

A cloned toy poodle produced from somatic cells derived from an aged female dog

To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.

Improved efficiency of canine nucleus transfer using roscovitine-treated canine fibroblasts

The aim of this study was to investigate whether roscovitine (the cyclin-dependent kinase 2 inhibitor) effectively induces synchronization of the donor cell cycle at G0/G1 and to examine the effect of donor cell cycle synchronization protocols on canine somatic cell nucleus transfer. Canine fibroblasts were obtained from skin biopsy cultures taken from a 7-yr-old retriever. The donor cell cycle was synchronized either by culturing cells to reach confluency or by treating cells with 15 microg/mL roscovitine for 24h. Cell cycle stages and apoptosis were analyzed by flow cytometry. After synchronization of the donor cell cycle, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into 18 naturally estrus-synchronized surrogates. There was no significant difference in cell cycle synchronization and apoptosis rates between the confluent and roscovitine groups. After transfer of reconstructed embryos, pregnancy was detected in three of nine surrogates that received cloned embryos reconstructed with roscovitine-treated cells, whereas only one of nine surrogates was pregnant after transfer of cloned embryos reconstructed with confluent cells. One pregnant female from the confluent cell group delivered one live and one dead pup, but the live one died within 5 days after birth. Three pregnant females from the roscovitine-treated cell group delivered eight live pups and one dead pup, and one of eight live pups died within 6 days after birth. In conclusion, the current results demonstrated that reconstructing embryos with roscovitine-treated cells resulted in increased efficiency of canine somatic cell nucleus transfer.

Recloned dogs derived from adipose stem cells of a transgenic cloned beagle

A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning.

Generation of transgenic dogs that conditionally express green fluorescent protein

We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet‐on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP‐positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies. genesis 49:472–478, 2011.