JungEun Park; HyunJu Oh; MinJung Kim; GeonA Kim; EunJung Park; YoungKwang Jo; Goo Jang; ByeongChun Lee
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, Korea
Cloning research is highlighted by the reason that it can be applied to many fields of biomedicine including xenotransplantation, production of bioreactor, cell therapy, and development of disease models for human.1,2 After the successful birth of the first cloned sheep, despite various attempts to improve and advance the cloning research, its efficiency is still low. Particularly in dog, it is harder to successfully produce a cloned puppy since dogs have unique reproductive physiology such as oocytes ovulation at GV stages.3,4 On the bright side, dogs (Canis familiaris), as men's best friends, are not only easy to handle and to communicate with, but also have many similar diseases to human being. Owing to these reasons, scientific necessity of cloned dogs has been raised, and eventually, the first cloned male afghan hounds, named "Snuppy", was born from adult ear skin fibroblast in 2005.5 Thereafter, several cloned canids including three female afghan hounds, a toy poodle, Pekingese, two beagles, two female and three male wolves, and Sapsaree, a Korean natural monument have been successfully produced by somatic cell nuclear transfer (SCNT).6-9 Recently, seven cloned drug sniffing dogs and four cancer sniffing dogs were produced by nuclear transfer from adult somatic donor cell collected from elite drug/cancer sniffing dogs.10 In order to produce human disease model animals, we produce transgenic red fluorescence (RFP) clone dog and inducible transgenic green fluorescence dog (GFP).11,12 All our research on canine SCNT can be applied to 1) conserving endangered canine species using inter-species SCNT, 2) developing the disease models for human, and 3) assisting human life by propagation of elite service dogs.
Production of Cloned Dogs From SCNT
In 2005, we produced the world's first cloned dog from adult somatic cells, named 'Snuppy', which means Seoul National University puppy.5 For dog cloning, the enucleated in vivo matured oocytes, which were collected by flushing the oviducts around 72 h after ovulation, were fused with a donor somatic cell derived from ear skin or fetal tissues. The reconstructed embryos were transferred into the oviducts of surrogate mothers and the cloned dogs were delivered by C-sec or natural delivery. For parental analysis, microsatellite analysis using genomic DNA from donor cells, cloned dogs, and surrogate mothers was carried out and hyper-variable regions of mitochondrial DNA (mtDNA) was sequenced to find out transmission of mtDNA in cloned dogs. Results showed that all clones were genetically identical to their donor cells, but their mtDNA had been derived from their oocyte donor dogs. After "Snuppy", we obtained three healthy female puppies cloned from somatic cells of a female Afghan hound.6 We have provided the first demonstration that female dogs can be produced by nuclear transfer of ear fibroblasts into enucleated canine oocytes. We also demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a 14 year-old female toy poodle and a 9 year-old female Pekingese, with enucleated in vivo matured oocytes of large-breed females, which were then transferred into the oviduct of large-breed recipient female dogs.13,14 Recently, seven cloned drug sniffing dogs were produced by nuclear transfer from roscovitine treated adult somatic cells.10 The present results demonstrated that reconstructing embryos with roscovitine treated cells results in increased efficiency in canine somatic cell nuclear transfer. Next, four cancer sniffing dogs were produced by nuclear transfer from adult somatic donor cells collected from elite cancer sniffing dogs. All clones were genetically identical to their donor cells, but mitochondrial DNA had originated from their oocyte donor dogs. These results indicate that SCNT has potential abilities of producing of dogs with elite capacity as service dogs. The successful rate was very low at time of the first cloned dog (3 pregnancies from 123 recipients); however, its rate has improved to around 15 to 30% by the improvement of canine SCNT.
Dog Cloning for Conservation of Endangered Canine Species
Recent progress in assisted reproductive techniques such as embryo cryopreservation, AI, and somatic cell nuclear transfer (SCNT), has demonstrated the techniques' potential application for maintaining endangered species. In particular, SCNT has been used to produce several kinds of somatic cell-cloned mammals. For this reason, it has promise for maintaining endangered species or restoring extinct species. The use of intraspecies and interspecies SCNT techniques has great potential as tools for the conservation of species with limited availability of oocytes and recipients. In 2007, as we produced cloned gray wolf (Canis lupus), an endangered species, the somatic cells were obtained from gray wolf and then transferred into in vivo-matured domestic dog oocytes. Consequently, cloned female wolf pups were produced and we demonstrated that SCNT is a practical approach for conserving endangered canids.8 Moreover, we demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells which were isolated from a postmortem wolf, then transferred into enucleated dog oocytes.9 Thus, SCNT has potential for preservation of canine species in extreme situations, including sudden death. In 2007, we produced two Sapsaree dog, a Korean natural monument, by nuclear transfer from adult somatic cells, once again demonstrating that canine SCNT can provide a practical approach for conserving endangered canine breeds.7
Dog Cloning for Animal Model Research
Dogs share a large number of disease types with humans2,15, and so have been considered prime medical research models. While gene targeting using embryonic stem (ES) cells has been the predominant procedure for generating transgenic mice models, application of this technology to the generation of large transgenic animals largely has not been available, mostly due to difficulty in obtaining the ES cells. Thus, cloning technology using SCNT has been regarded as an alternative approach for gene targeting in large animals.16,17 In application of SCNT technology as an alternative approach of gene targeting in dogs, fetal fibroblasts were used in generating gene engineered donor cell because of their nature to grow fast and ability to maintain their characteristics before going through cellular senescence.16,18,19 In an attempt to perform dog SCNT using fetal fibroblast as a donor cell, the beneficial effects of fetal fibroblast in fusion rate as well as in developmental competence to cloned offspring were identified.20 Based on these results, canine fetal fibroblasts were used in place of ES cells to obtain cell lines with RFP transgene insertion in their genome, and SCNT technique was employed in producing cloned transgenic dogs.11 We confirmed the integration, transcription, and expression of RFP gene in all cloned puppies with RT-PCR, Southern blot, and detection of RFP fluorescence. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP.
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.21,22 As alternative for fetal fibroblast, we used adult stem cells as nuclear donors in canine SCNT. We performed 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 this study, we demonstrated that cAd-MSCs from cloned transgenic dogs have the capacity to differentiate into mesodermal and ectodermal lineages in vitro, and that cAd-MSCs can be used to generate cloned pups by SCNT, for the first time.23 Furthermore, we have confirmed that recloning using cAd-MSCs is capable of producing multiple genetic modified clones, and that utilization of cAd-MSCs may prove to be an excellent cell type for producing a genetic disease model.
For the next step, we generated transgenic dogs by employing an inducible gene expression approach to overcome the problems of uncontrollable expression of exogenous transgenes.12 As a result, fetal fibroblasts infected with a Tet-on eGFP vector were used for SCNT, and we successfully produced the transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. These advancements in canine SCNT will contribute to expanding the scope of canine SCNT application and producing canine transgenic models for research in human and veterinary medicine or biomedical science.
Progress in canine SCNT has been very slow compared to in other mammalian species and many technical difficulties still lie ahead. However, since the birth of the first cloned dog, SCNT in canines has been lauded as a major scientific breakthrough for both animal and human medical science. Our laboratory successfully produced several cloned canine offspring to expand the pool of elite service dogs (rescue, drug sniffing, guiding the blind and deaf, etc.), to conserve endangered canine species, and to aid in the development of animal models for human disease. Taken together, the established procedure of the dog cloning and production of transgenic dog from this study shall open a new door to the generation of new transgenic dogs and their contribution to the future biomedical research.
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