Dolly at 25… is she ‘… still goin’ strong?’

The birth of ‘Dolly the sheep’ (the first animal to be cloned from an adult (somatic) cell) on 5th July 1996 marked a seminal moment in the field of developmental genetics for several reasons. It first provided unequivocal evidence of genomic equivalence between embryonic and somatic cells by demonstrating that it is possible to re-establish a pluripotent state in differentiated cells. In so doing, it inspired others to later identify at least some of the factors required for the induction of pluripotency in cultured somatic cells leading to the creation of ‘induced’ pluripotent stem cells (iPSCs) for therapeutic applications in both human and animal medicine (Takahashi & Yamanaka 2006). However, it was the need to develop an effective method by which to produce genetically modified (GM) livestock that drove the programme of work that ultimately led to the birth of Dolly. Existing GM approaches at that time relied on techniques, such as pronuclear DNA microinjection. These techniques were both inefficient and limited in their ability to introduce genetic change in large animals. Furthermore, in contrast to mice (Evans & Kaufman 1981, Martin 1981), the derivation and establishment of germ-line competent embryonic-stem cells (so amenable for introducing genetic modification in that species) has thus far remained elusive in livestock species. Thus, the approach adopted by Wilmut et al. (1997) involved the transfer of nuclei from somatic (i.e. cultured mammary) cells to enucleated oocytes followed by electrofusion and activation; a procedure referred to as somatic-cell nuclear transfer (SCNT). The success of Dolly led quickly to a succession of papers reporting the birth of GM livestock derived from genetically altered somatic cells (e.g. Schnieke et al. 1997, Cibelli et al. 1998, Dai et al. 2002). However, both ethical and biological concerns regarding the underlying cloning procedure were raised at the time. Biological concerns related to the overall efficiency of SCNT, in utero and perinatal losses, together with the ‘biological’ age, longevity and health of cloned offspring; all of which threatened the prospect of generating GM animals by this means. In utero losses, developmental abnormalities and neonatal morbidity were attributable, at least in part, to the culture of gametes and embryos, which contribute independently to epigenetic dysregulation at both imprinted and non-imprinted loci (Young et al. 2001, Chen et al. 2017), leading to a phenomenon known as the large offspring syndrome (Young et al. 1998). Concerns were raised specifically about the health of Dolly, as she was diagnosed with osteoarthritis (OA) of the left stifle at a relatively young (51⁄2 years) age (Rhind et al. 2004). It was suggested that she might have aged prematurely, and terminal fragment restriction analyses of her genomic DNA appeared to support the concept of telomere shortening (Shiels et al. 1999). However, this observation was at odds with those from other SCNT studies which generally found telomeres to ‘rejuvenate’ during nuclear reprogramming (Marión & Blasco 2010). Furthermore, our own retrospective radiographic assessments of the skeletons of Dolly, Megan and Morag (the latter two sheep had been cloned previously from differentiated cells; Campbell et al. 1996) reported a prevalence and severity of OA no different to that of naturally conceived sheep of comparable age (Corr et al. 2017). Indeed, several studies over the years have concluded that cloned offspring which survive beyond the neonatal period are healthy, age normally, produce viable offspring and animal products safe for human consumption (Lanza et al. 2001, Yang et al. 2007, Watanabe 2013, Sinclair et al. 2016). Yet concerns relating to animal welfare remain, and these have been sufficient to enforce a ban on commercial farm-animal cloning within the UK and EU, although not within the US and many other countries. The consequence of this ban is that, in an era of comparatively well-funded iPSC research, the ability to undertake studies to improve the overall efficiency, safety and application of SCNT has been impeded. Yet, despite the global investment in iPSC research over the last 20 years, its potential for cell-based therapy has yet to be realised (Yamanaka 2020). Other commentators emphasise the merits of zygotic genome editing, using designer nucleases such as CRISPR/Cas9, to generate GM