Parkinsonian dogs

Cloned edited beagles were born in South Korea

Polina Loseva, N+1

Researchers from South Korea have obtained two beagle clones with a mutation likely associated with Parkinson's disease. To do this, it was necessary to first edit the dog's cells, and then create embryos from them. Two embryos have grown into full-fledged healthy puppies that carry the desired mutation, and also glow in ultraviolet. The work was published in the journal BMC Biotechnology (Kim et al., Generation of genome-edited dogs by somatic cell nuclear transfer).

Born puppies glow green, unlike the control beagle.

Dogs often become a platform for testing genetic technologies. On the one hand, they are closer to humans in a number of parameters than mice, and it is easier to model a number of diseases on them. On the other hand, genetic engineering allows faster than breeding, to output lines with the right properties — which are in demand not only in science, but also, for example, as guards or bloodhounds.

In 2005, the dog was cloned for the first time. In 2015, it was edited with CRISPR/Cas9 to build more muscle mass. At the next stage, it was necessary to combine these two methods in order to reproduce the qualities of the original individual and add new properties to them. Chinese scientists did this in 2018, but in two stages, or rather in two generations. First, they edited the fertilized zygotes of dogs, introducing mutations into the ApoE genomic locus — it is associated, among other things, with the transport of cholesterol in the blood and the risk of developing Alzheimer's disease (in humans). And then, when the ApoE mutant puppies were born, fibroblasts from the connective tissues of the ear were taken from them and their nuclei were transplanted into donor eggs. As a result, in the third generation, clones with mutations in the right part of the DNA turned out.

Now researchers from South Korea have tried to simplify this method in order to achieve results already in the first generation. A group of scientists from the National University of Chunnam under the leadership of Min-Kyu Kim (Min-Kyu Kim) decided to do without the intermediate stage. This allowed it would also speed up the process, since the breeding cycle in dogs is quite long. Geneticists undertook to create animals with a mutation in the DJ-1 gene. It is believed that it may be somehow involved in the development of Parkinson's disease - and it is better to model it on dogs than on mice.

Scientists took a sample of fibroblasts from a beagle embryo and injected into these cells a vector containing all the necessary components for CRISPR/Cas9 to work. In addition, this vector carried a neomycin resistance gene and a green fluorescent protein gene. These inserts were needed to follow the fate of the edited cells. After editing, the fibroblast culture was treated with neomycin — and the cells that the vector did not hit died. The remaining ones glowed green in the ultraviolet — thus, the vector was inside them and worked, producing proteins (which means that the CRISPR/Cas9 system should also work inside).

After that, geneticists took the eggs of dogs, removed the nuclei from them and, applying a current to them, forced them to merge with the edited fibroblasts. In total, 68 single-celled embryos turned out, which were planted in six females. Only one of them got pregnant: she had two embryos, and later two beagle puppies were born. Both of them glow in ultraviolet and do not produce DJ-1 protein. At least up to 14 months (by the time of publication of the article), they developed normally and were healthy. To find out whether they will develop symptoms similar to Parkinson's disease, the authors of the work plan to monitor them further.

The researchers suggest that such a method can be used further to support bred breeds or create models for diseases. However, they emphasize that their method is not perfect: they used a vector that is embedded in the cellular genome. This means that CRISPR/Cas9 can work for a long time inside cells and make unnecessary cuts in DNA. The authors of the work checked the genome of the edited beagles and found no off-target mutations in the places where they expected. And yet they admit that it would be safer to use non-embedding vectors or introduce the editing system molecules themselves into the cell.

Not all attempts to edit dogs are aimed at causing them to get sick — some scientists, on the contrary, practice therapy methods on them: for example, they once tried using CRISPR/Cas9 to cure dogs of Duchenne myodystrophy.

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