Characterization of Myostatin Gene-Edited Pigs

Since discovery of the regulatory role of myostatin in muscle development, there have been a number of reports demonstrating the effect of polymorphisms in the myostatin gene (MSTN) across various species. In particular, naturally occurring mutations that increase muscle growth have been reported in mice, cattle, man, dogs, horses and sheep. Conversely, investigation of MSTN polymorphisms in pigs has not been as fruitful with no true loss-of-function mutant, similar to those in other species, being identified.  It is possible that there has been insufficient screening of pig populations, however, it is also possible there is a biological restriction on the survivability of novel MSTN mutant genotypes. To investigate the potential consequences and utility of loss-of-function mutations in the pig, as well as possible interactions with known IGF2 polymorphisms, we have used zinc finger nucleases designed specifically to target key functional domains in the third exon of MSTN. We have successfully introduced a series of small insertion-deletion mutations in porcine fetal fibroblast cell lines.   Clonally isolated sub-lines were used for somatic cell nuclear transfer to successfully produce live monoallelic edited pigs.  We further investigated potential off-target modifications by complete genome sequencing of the fibroblast cell line prior to editing and a live-born edited clone.  Resulting variant calling suggests off target editing events are difficult to detect in the context of sequence variation due to technical precision of NGS platforms and potential somatic mutation. Selected matings are currently ongoing to investigate the effects of this mutation on muscle development.