Bulk RNA-seq analysis of wildtype and MYF5/MYOD/MYF6 knockdown porcine embryo
The pig is an important animal model that is increasingly used for biomedical research.  For example, pigs produced using somatic cell nuclear transfer (SCNT) technology are relevant for transplantation strategies either involving xenotransplantation or the development of human organs in pig for exotransplantation. Pigs, however, are less well characterized than other animal models such as rodents. In previous studies, we produced pigs that lacked skeletal muscle as this was the first step in humanizing this lineage, and we characterized these MYF5/MYOD/MYF6 knockdown embryos to E35. In the current studies, we have evaluated the development and myogenesis in mid-stage porcine embryos produced by SCNT (E41-E90). We comprehensively examined mid-stage MYF5/MYOD/MYF6 knockdown pigs produced by deleting MYF5/MYOD/MYF6 from porcine fibroblasts, cloning these fibroblasts, transferring the early embryos to surrogate gilts, and harvesting the resulting fetal pigs at defined gestational ages. Here, we observed that these genetic deletions resulted in a complete absence of skeletal muscle in these embryos and fetal pigs, an absence of ribs, vertebral abnormalities and an absence of a sternum. Importantly, we observed that these embryos failed to progress beyond E62 indicating embryonic lethality. In addition, we examined porcine myogenesis by evaluating wildtype comparators using gross anatomical, morphological, histological, molecular biological analyzes as well as magnetic resonance imaging of both WT and knockdown embryos and fetal pigs between the ages of E41-E90. We have determined that the knockdown phenotype in pigs is more severe than that observed in rodents as mice with this multiplexed deletion were lethal in the immediate postnatal period. These studies provide an important platform for engineering humanized muscle in gene edited pigs. Overall design: Limb muscle tissue form wild-type and MYF5/MYOD/MYF6 knockout E41 and E62 pig embryonic stages.