22 Establishment, characterisation, and validation of novel porcine embryonic fibroblasts as a potential source for genetic modification

Fibroblasts are a widely used cell type for somatic cell nuclear transfer (SCNT). In this report, we demonstrate the successful derivation of embryonic fibroblasts (EFs) from a blastocyst outgrowth culture that shares many characteristics with fetal fibroblasts (FF), including the spindle-shaped morphology and expression of the key fibroblast-specific molecular markers, VIMENTIN, and α-smooth muscle actin. Transcriptomic analysis via RNA sequencing further confirmed the close similarity of EF to FF. We performed SCNT using eGFP-labelled EFs and the founder FFs, resulting in enhanced in vivo developmental competence. Finally, we demonstrated the feasibility of establishing EFs from edited embryos and the feasibility of genetic modification directly in the EFs. By pre-screening, we identified 11.6% homology-directed repair (HDR) and 67.7% nonhomologous end joining (NHEJ) events within the EFs, and 10.8% HDR and 71.7% NHEJ events within the FFs, respectively. The overall editing rates in EFs were similar to that of FFs (n = 3, P < 0.01). The clonal lines for targeted mutations without the need to perform embryo transfers to establish FFs will potentially overcome the inherent disadvantages of the zygotic microinjection, including but not limited to the unpredictability of edited outcomes and mosaicism. The established EFs may, therefore, represent an important cell resource for gene editing and generating genetically modified animals.