Animal cloning has been popularized for more than two decades, since the birth of Dolly the Sheep 25 years ago in 1996. There has been an apparent waning of interest in cloning, evident by a reduced number of reports.

Over 1,500 dogs, representing ~20% of the American Kennel Club’s recognized breeds, have now been cloned, making the dog (Canis familiaris) one of the most successfully cloned mammals. Dogs have a unique relationship with humans, dating to prehistory, and a high degree of genome homology to humans.

A number of phenotypic variations, rarely recorded in natural reproduction have been observed in these more than 1,000 clones. These observations differ between donors and their clones, and between clones from the same donor, indicating a non-genetic effect. These differences cannot be fully explained by current understandings but point to epigenetic and cellular reprogramming effects of somatic cell nuclear transfer.

Notably, some phenotypic variations have been reversed through further cloning. Here we summarize these observations and elaborate on the cloning procedure.

…The first canine was cloned in 2005 and was the 15^th animal to be cloned (Figure 1c)¹. Unlike other species, canine cloning remains comparatively difficult, due to the lack of in vitro oocyte maturation methods and other reproductive complexities². Of the larger animals only a few, such as pigs and cattle have been reported to be cloned at a scale in the hundreds³.

The observations, reported herein, are based on more than 1,000 cloned dogs [of 1,500 total], produced at the [Sooam] center over the last decade. This success is attributed to a streamlining and combination of factors, including the optimization of established techniques. To date, a total of ~20% of recognized dog breeds have been cloned, by us, and are reported in this study. [ie. >5,000 dog clones total, including ViaGen, Sinogene etc.]

In spite of the difficulties related to the unique canine reproductive physiology, we have observed that factors thought to negatively influence cloning success may not be as vital as initially presumed. These factors include, among others, the breed of the cell donor, oocyte donor and surrogate, donor age and cell passage number. In fact, our data indicate a higher canine cloning efficiency than reported in most other commonly cloned species⁴.

The larger number and diversity of breeds seen in dogs surpasses that of any other animal species⁵. This has been considered being a barrier for canine reproduction between breeds. Our observations indicate that these barriers may be fewer and narrower in scope than previously assumed⁶.

…Cloning efficiency: Although it has been suggested that phylogenetic distance between oocyte donor, cell donor and surrogate plays a major role in cloning efficiency, calculated as the number of offspring per reconstructed oocytes⁴, we found no statistically-significant difference in the cloning efficiency of cell donors between distantly related canine breeds¹⁷, although there was a large degree of variation (Figure 2b).

We observed that the greatest cloning efficiencies occurred in individuals with extreme distances between breeds, however the average cloning efficiency of these “breeds did not differ statistically-significantly from the norm”¹⁸. This suggests that there is a greater contribution to cloning efficiency from individual cell donors than the differences associated between breeds (Figure 3). We report canine cloning efficiency, based on the number of live offspring produced from the number of transferred reconstructed oocytes, to be above 2.0% (Figure 3, Supplemental Figure 1; Supplemental Table 2) which is moderately higher than that of most other reported species¹⁹.

…No differences in interbreed efficiency, with sufficient sample size, is indicated by greater genetic distance within the domestic dog and related species, such as the coyote and wolf on a modified cladogram from¹⁷ (Figure 3b). More genetically distant canine species resulted in a failure of the technique. This was observed when clones of the African wild dog (Lycaon pictus) failed to go to term, although initial pregnancy rates were similar⁹. Other more dissimilar species, such as the red fox (Vulpes vulpes) and the raccoon dog (Nyctereutes procyonoides) did not result in any detectable pregnancies (Figure 1a).

…Postnatal survival of cloned dogs: …Health assessment has been performed using a modified APGAR scoring method, capable of characterizing pup survival rates with accuracy. At this point adult survival is next to guaranteed in cloned pups which are born healthy. Apart from a few cases of care-related deaths (<2%), generally attributed to the surrogate mothers, there have been no premature death or increased incidence of disease observed or reported in cloned dogs, assessed to be healthy at birth. The longevity of cloned dogs does not appear to be diminished compared to breed averages, based on observations to date. The oldest living cloned animal, as far as we know, is a cloned dog, residing in Korea, born in March 2007.

…Phenotypic variations in cloned dogs: The cloning process allows for the observation of multiple genetically identical individuals, which may shed light on various developmental associated phenotypes. These cases may warrant further investigation into similar human conditions as they relate to the phenomena observed in cloned dogs. Occasionally phenotypic variations are observed from genetically identical individuals following the cloning process²⁹. Here we describe several such observations from individuals from the 1,000+ clones produced over the last 10+ years (Supplemental Table 1; Supplemental Figure 3).

…It is thought that these peculiarities in the cloning process are due to incomplete cellular reprogramming, resulting from the methylation or genetic regulated state of the donor cell⁴⁶. Cell type and origin, of the donor, may be associated with these formations⁴⁷. We agree that the evidence shows an epigenetic mechanism associated with the reprogramming process as described above, treatment with the methylation inhibitor, AZA, reduced phenotypic abnormalities when administered prior to SCNT.

…Conclusion: Canine cloning has matured over the last decade, in part due to refinement of cloning techniques. A relatively low number of phenotypic variations have been reported. No abnormalities related to longevity of healthy born clones have been identified. Future potential applications of animal cloning continues to have the potential for reproductive rescue of endangered and extinct species. Cloned animals may additionally provide information for human medical insights. The successful cloning of dogs in increasing number has indicated the increased general interest regarding cloning and the general outlook of cloning appears to be shifting towards acceptance. Several practical and ethical questions persist and should be continually evaluated with consideration for both advantages and disadvantages related to the various use of animals. Cloning in the technological toolkit of biology will undoubtedly continue to play a role in the production and conservation of canids and other species into the future.

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