By Sara Su, Animal Science and English ’24

INTRODUCTION

In 1996, Dolly the sheep was the first mammal to be successfully cloned [1, 2]. Since then, 22 other animal species have been cloned, including rats, mice, cattle, goats, camels, cats, pigs, mules, and horses [3-12]. Among these, about 19 species have clones surviving to adulthood. In 2005, the first cloned dog to survive to adulthood was named Snuppy, who was derived from somatic cells from the ear-skin of a male Afghan hound. He was the 15th animal to be cloned and lived to the age of 10 [13]. He was cloned using Somatic Cell Nuclear Transfer (SCNT), a common method that involves removing the nucleus from an oocyte (egg cell) and replacing it with a nucleus from a somatic cell, typically a fibroblast [14].  Fibroblasts are a type of stromal cell found in connective tissues such as the skin and tendons – they are often used in cloning because they are relatively easy to culture. After nuclear transfer, this reconstructed oocyte, which is similar to a fertilized egg, is then activated and transferred into the oviduct of a surrogate female, usually in groups of 10-15 cells. After pregnancy is confirmed, viable offspring are born via C-Section [13-16]. Though SCNT is the most viable method of cloning so far, it remains very inefficient and the live birth ratio is extremely low [13, 17, 18]. Nearly 30 years after Dolly, little is understood about cloning, which presents unique challenges to different species; this review will discuss the relevance of the canine model in regards to human health, canine-specific challenges in using SCNT for cloning, as well as recent trends among successfully cloned dogs.

The Interest in Dogs as a Medical Model

Overall, dogs have become increasingly relevant as a medical model for human diseases in the 21st century. This is because many heritable canine diseases are orthological to human ones, which means they share similar traits and functions [19]. The dog genome is found to be closer to the human genome than the mouse genome – while mice are commonly used as medical models for humans, canine models have also proven to be useful for comparative studies due to their relatively long life, larger size, and similar tissue functions[20]. There are at least 350 shared genetic diseases between dogs and humans discovered so far, affecting a variety of systems such as the dermatological, lysosomal, hematological/immunological, and muscular/skeletal systems [21]. All of these contribute to the rising application of canine medical models to study disease mechanisms for well-known conditions such as Alzheimers and diabetes, while also being able to explore clinical therapies for rare genetic diseases that would otherwise be difficult to study. Other fields of study using canine colonies include but are not limited to: organ transplants, drug development, non-invasive biomarker generation, and psychological disorders [19-24].

Dog-Specific Challenges in Cloning

There are a few species-specific reasons why dog cloning remains inefficient. Cloning efficiency, defined as the ratio of live offspring coming from the number of transferred reconstructed oocytes, is usually not more than 3% across all species, regardless of the age or type of donor cell. Additionally, the average cloning efficiency between breeds does not differ significantly [21]. For canines, cloning efficiency is higher than many other reported species, at 2% [25]. However, this is still an extremely low number, and a specific challenge when it comes to dogs is the viable maturation of oocytes in vitro [13, 26]. It should be noted that the pregnancy rate can be increased by increasing the number of reconstructed oocytes injected into surrogates, but cloning efficiency itself is not changed. Another issue with canines is the vast number of breeds within the species – it is difficult to select for compatible nuclear/oocyte donors, in addition to adequate surrogate selection [21]. Finally, a widespread issue with cloned individuals is postnatal care – although survival is pretty much guaranteed for clones that are born healthy, cloned animals are just as vulnerable to disease and poor management as any other species [21]. Dolly the sheep died early from such an instance, rather than complications directly related to cloning. Overall, there is insufficient knowledge of the nuances of canine reproductive systems, including a lack of comprehensive protocols regarding oocyte maturation in culture and specific methods of post-natal clone care, leading to further difficulties in dog-specific cloning. 

Snuppy and His Clones

As previously stated, the first dog to be successfully cloned and survive to adulthood was a male Afghan hound named Snuppy, short for Seoul National University Puppy. Snuppy was born in 2005, and was the only survivor out of 123 recipient surrogates. Snuppy was cloned from fibroblast cultures derived from the biopsy of the ear-skin of an Afghan hound named Tai. He was confirmed to be genetically identical to Tai through the use of canine-specific biomarkers [27]. For this experiment, 3 out of 123 surrogates resulted in pregnancies, 2 were carried to term, and 1 survived to adulthood – the other puppy died on day 22 due to aspiration pneumonia after experiencing neonatal respiratory distress. Although the efficiency of cloning is very low in the first place, this particular experiment had a cloning efficiency rate much lower than expected – 2 puppies were born to 123 surrogates, or 1.6% [13]. Snuppy ended up living to be 10 years old, while his donor Tai lived to be 12 years old – both individuals died of cancer-related causes, but were generally healthy until then. It should be noted that the median lifespan of Afghan hounds is reported to be 11.9 years, so their lifespans were not out of the norm [28].

In 2017, Snuppy was cloned. This time the cloning efficiency and success rates were much higher and resulted in 3 clones who are still alive today. Rather than using fibroblasts, this experiment used adipose-derived mesenchymal stem cells (ASCs). Then, ASCs were cultured with Dulbecco’s Modified Eagle Medium(MDEM), a technique that increases oocyte fusion rate in SCNT[29]. This experiment resulted in pregnancy and delivery rates of 42.9% (3 dogs out of 7 recipients) and 4.3% (4 clones out of 94 embryos). Compared to Snuppy’s 2.4% (3 out of 123) and 0.2% (2 from 1,095), these changes in technique correlated in a huge jump in overall efficiency [13, 28]. 

Other studies have also been published exploring the viability of cloned working dogs. For dogs, SCNT can be used regardless of sex, age, and breed [13, 30]. It was recently concluded that cloned dogs have similar behavior patterns to their cell donors, and can lead healthy lives with life spans comparable to naturally bred dogs [21, 29]. Overall, about 20% of dog breeds recognized by the American Kennel Club have been successfully cloned, which is highly successful compared to other mammals [21]. Though more research is needed to improve dog cloning efficiency, it has already been proven that clones of drug detection dogs[31] and cancer-sniffing dogs[33] outperform naturally bred dogs, scoring higher averages on qualification tests for these services [32-33].

CONCLUSION

To conclude, both studies regarding the creation of Snuppy and the subsequent cloning of his cells demonstrate great potential for the common use of canine clones in the modern world. Multiple obstacles regarding canine cloning were recognized and overcome, though the cloning efficiency rate can be further improved by obtaining greater knowledge of the canine reproductive system. Additionally, it was proven that clones who are born healthy aren’t at a larger risk for diseases or a shortened lifespan – they are comparable to the average puppy. All of this contributes to the feasibility of cloning working dogs – studies are already exploring the possibility of using clones of dogs that perform drug- and disease- detection, knowing that the physical qualifications for such jobs are strongly linked to specific genetic traits.

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