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 humans^2,15, 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.

Conclusions

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.

References

1.  Ostrander EA, Galibert F, Patterson DF. Canine genetics comes of age. Trends Genet 2000;16:117–124.

2.  Mack GS. Cancer researchers usher in dog days of medicine. Nat Med 2005;11:1018.

3.  Rodrigues BA, Rodrigues JL. Meiotic response of in vitro matured canine oocytes under different proteins and heterologous hormone supplementation. Reprod Domest Anim 2003;38:58–62.

4.  Hossein MS, Kim MK, Jang G, et al. Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages. Mol Reprod Dev 2007;74:1213–1220.

5.  Lee BC, Kim MK, Jang G, et al. Dogs cloned from adult somatic cells. Nature 2005;436:641.

6.  Jang G, Kim MK, Oh HJ, et al. Birth of viable female dogs produced by somatic cell nuclear transfer. Theriogenology 2007;67:941–947.

7.  Jang G, Hong S, Kang J, et al. Conservation of the Sapsaree (Canis familiaris), a Korean natural monument, using somatic cell nuclear transfer. J Vet Med Sci 2009;71:1217–1220.

8.  Kim MK, Jang G, Oh HJ, et al. Endangered wolves cloned from adult somatic cells. Cloning Stem Cells 2007;9:130–137.

9.  Oh HJ, Kim MK, Jang G, et al. Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem. Theriogenology 2008;70:638–647.

10. Oh HJ, Hong SG, Park JE, et al. Improved efficiency of canine nucleus transfer using roscovitine-treated canine fibroblasts. Theriogenology 2009;72:461–470.

11. Hong SG, Kim MK, Jang G, et al. Generation of red fluorescent protein transgenic dogs. Genesis 2009;47:314–322.

12. Kim MJ, Oh HJ, Park JE, et al. Generation of transgenic dogs that conditionally express green fluorescent protein. Genesis 2011;49:472–478.

13. Jang G, Hong SG, Oh HJ, et al. A cloned toy poodle produced from somatic cells derived from an aged female dog. Theriogenology 2008;69:556–563.

14. Park J, Oh H, Hong S, et al. Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer. Theriogenology 2011;75:777–782.

15. Sutter NB, Ostrander EA. Dog star rising: the canine genetic system. Nat Rev Genet 2004;5:900–910.

16. McCreath KJ, Howcroft J, Campbell KH, et al. Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 2000;405:1066–1069.

17. Denning C, Priddle H. New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells. Reproduction 2003;126:1–11.

18. Schnieke AE, Kind AJ, Ritchie WA, et al. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science 1997;278:2130–2133.

19. Cibelli JB, Stice SL, Golueke PJ, et al. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 1998;280:1256–1258.

20. Hong SG, Jang G, Kim MK, et al. Dogs cloned from fetal fibroblasts by nuclear transfer. Anim Reprod Sci 2009;115:334–339.

21. Eggan K, Rode A, Jentsch I, et al. Male and female mice derived from the same embryonic stem cell clone by tetraploid embryo complementation. Nat Biotechnol 2002;20:455–459.

22. Jeanisch R, Eggan K, Humpherys D, et al. Nuclear cloning, stem cells, and genomic reprogramming. Cloning Stem Cells 2002;4:389–396.

23. Oh HJ, Park JE, Kim MJ, et al. Recloned dogs derived from adipose stem cells of a transgenic cloned beagle. Theriogenology 2011;75:1221–1231.