Gene-Editing of Porcine Myostatin using Zinc-Finger Nucleases

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 (MSTN) gene 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 myostatin polymorphisms in pigs has not been as fruitful with no true loss-of-function mutant, similar to those in other species, being identified. As commercial pig breeding populations are under relatively high selection intensity for lean yield, the failure to detect spontaneous mutations in pig myostatin that significantly increase muscularity would seem curious given the observations from other species. 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 myostatin mutant genotypes, particularly loss-of-function mutations.  To investigate the potential consequences and utility of loss-of-function mutations in the pig, we have used zinc finger nucleases (ZFN) designed specifically to target key functional domains in the third exon of MSTN.  Zinc finger nucleases allow targeted editing of the genome by creating double-stranded DNA (dsDNA) breaks at specified locations.  To date, we have successfully introduced a series of small insertion-deletion mutations in porcine fetal fibroblast cell lines derived from both a male and female individuals.   Clonally isolated sub-lines are being prepared for somatic cell nuclear transfer.