Lessons Learned from Cloning Dogs

Conservation of endangered species

The number of species on the Species Survival Commission’s Red List is increasing because of human activities. Of the 5487 mammal species, nearly one-quarter (1219) are globally threatened or extinct (http://www.iucnredlist.org/). To maintain genetic biodiversity and reduce the interval between generations, ARTs for endangered species have been developed (Pukazhenthi et al. 2005). Cloning has the potential to be a powerful tool for preservation of endangered animals. The first successful cloning attempt in endangered canids was the gray wolf (Canis lupus) that is considered a threatened species in many countries (Kim et al. 2007b). Because of limited availability of oocytes and recipients, interspecies SCNT has been used to clone the endangered animals, using a wolf somatic cell and a dog oocyte and recipient (Kim et al. 2007b; Oh et al. 2008). The mitochondrial (mt) DNA sequences in the cloned wolf offspring were identical to those of the oocyte donor dog, in agreement with results in a cloned gaur (Lanza et al. 2000) and mouflon (Loi et al. 2001). Furthermore, a fibroblast derived from a gray wolf 6 h after death was also successfully cloned (Oh et al. 2008). Therefore, interspecies SCNT in dogs can be used for preservation of endangered canine species, both within a conservation programme and also in extreme situations involving sudden death. Two clones of the Sapsaree breed, which is registered as a national monument in South Korea, were produced using mixed breed dog oocytes (Jang et al. 2009). The clones were genetically identical to the nuclear donor dog, but their mtDNA originated from the oocyte donor dogs (Jang et al. 2009). Thus, if a female clone breeds with a wild-type male, the mtDNA of their progeny would be derived from the oocyte donor dog. In contrast, although cloned males obtained their mtDNA from oocyte donor dogs, the mtDNA of their sperm is not transmitted to their progeny, indicating that genetic characteristics of an individual can pass to the second generation. Therefore, cloning of male endangered species can be recommended for conservation programme.

Cloning of pet dogs

The reproductive ability of dogs is terminated or impaired due to removal of the reproductive organs or due to age. Removal of a dog’s reproductive organs may be indicated because of disease, to prevent further diseases, or most commonly to prevent breeding. More than 80% of sexually intact male dogs >5 years old had benign prostatic hypertrophy, and in most cases, castration is a recommended treatment (Memon 2007). Even in healthy older dogs, pregnancy is not recommended. Primipara >6 years old had low reproductive ability, because of special obstetric conditions and stillbirths compared with young primipara (Munnich and Kuchenmeister 2009). Thus, it is difficult to propagate neutered or old dogs with impaired reproductive ability using conventional methods. As an alternative, cloning can restore reproductive ability in such cases (Jang et al. 2008; Park et al. 2009b). A 14-year-old toy poodle was cloned using a large breed dog as a recipient (Jang et al. 2008). Telomere lengths of the clone were not significantly different from those of the aged donor dog or the age-matched control dog (Jang et al. 2008). In addition, a dead pet dog can also be reproduced by SCNT, if some cells are maintained permanently by cryopreservation to preserve chromosome stability and genetic integrity. The first commercially cloned pet dogs were five pit bull terriers produced in 2008, 3 years after the cell donor had died (http://news.bbc.co.uk/2/hi/7542338.stm).

Propagation of elite dogs

Most of the genetic information, except mt DNA, is transmitted from a somatic cell donor to a clone. Although some differences can be seen in phenotypes of cloned animals, especially related to epigenetic reprogramming (Schmidt-Kuntzel et al. 2009), characteristics related to nuclear genetic information can be passed on to the clones, so that a clone could inherit elite ability from the cell donor if the related gene is naturally present or inserted into the donor’s genome. Because re-cloned dogs have been obtained, theoretically, infinite propagation of the elite ability is also possible. Therefore, cloning can be a powerful tool to produce dogs with an excellent talent identical to that of their donors because an elite dog’s abilities cannot be passed on to the next generation via other ARTs. For example, it is believed that the scent sniffing ability of a dog originates from the anatomical structure of the nasal cavity (Craven et al. 2007) or from gene expression in olfactory receptors (Robin et al. 2009). In a cancer-sniffing dog, the sensitivity of scent detection in stool samples from patients with colorectal cancer compared with conventional diagnosis by colonoscopy was 0.97 and the specificity was 0.99 (Sonoda et al. 2011). The accuracy of canine scent detection was high even for early cancer (Sonoda et al. 2011). To propagate the cancer-sniffing dog, SCNT technology was applied. Successful cloning of a cancer-sniffing dog was reported, and four clones were generated from a female neutered dog; the clones are growing and healthy without apparent abnormalities (Park et al. 2009b). To evaluate the success of cloning technology for propagation of the cancer-sniffing dogs, the sniffing talent in the cloned dogs is currently being tested and compared to the donor dogs; the results will be reported after a complete evaluation.

Production of transgenic cloned dogs

Dogs have been used as animal models for human diseases because of the similarities in their size, longevity and physiology to humans. Of the nearly 580 known hereditary diseases described in the dog, more than half (288) can be potential models for human diseases (http://omia.angis.org.au, accessed May 2012). Because production of transgenic large animals using pronucleus injection or homologous recombination in embryonic stem cells is not available, recent successes in creating transgenic animals (Schnieke et al. 1997; Cibelli et al. 1998; Dai et al. 2002) using SCNT give promise to future generation of genetically modified models in large animals including dogs.

Up until now, two kinds of transgenic dogs expressing red fluorescent protein (RFP) genes constitutively (Hong et al. 2009b) and green fluorescent protein (GFP) genes conditionally (Kim et al. 2011) have been reported. All the dogs were delivered healthy at birth except two: one pup died at day 10 after birth because of interstitial pneumonia (Kim et al. 2011) and the other died because of chronic bronchopneumonia at 11 weeks after birth (Hong et al. 2011b). Regarding the death of the transgenic pups, it is difficult to conclude that the integration of transgenes into their genome was the cause of the health problems because the death frequency of cloned pups is within the reference range (Johnston et al. 2001). In addition, transgenic dogs born by SCNT exhibited normal reproductive ability, and the exogeneous gene was successfully transmitted to their progeny as well as stable insertion of the transgene into the genome (Hong et al. 2011c; Kim et al. 2011). Therefore, the previous results indicate that SCNT technology in dogs can be applied to produce transgenic dogs.

On the basis of the previous successful cloning of RFP or GFP transgenic dogs, we are now producing transgenic dogs for application to the neuronal degenerative Alzheimer’s disease (AD). Although the pathogenesis of AD has been a subject of intensive research for the last several decades worldwide, treatment or preventive measures have so far produced no breakthrough because of the lack of appropriate AD models. Mouse models have a limitation for AD research because the irreconcilable species gap between rodents and humans has impeded the research itself as well as the translation of findings from rodent studies to human cases (Kokjohn and Roher, 2009). As an alternative to the mouse model, we performed a preliminary study to produce a transgenic dog that expresses a neuron-specific RFP transgene in brain by SCNT. A transgenic dog expressing the RFP transgene in neural cells of the brain under human synapsin 1 promoter was generated (Oh et al. 2012), indicating that the human synapsin promoter is functional. Currently, we are performing a study to produce a neural-specific transgene expressing dog, which will be a pivotal animal model for neurodegenerative AD.