Episomal Reprogramming of Equine Fibroblasts

Equine fibroblasts were transfected with different combinations of episomal reprogramming plasmids that have demonstrated to reprogram human cells [38 (link)–40 (link)]: pCXLE-hOCT3/4-shp53-F (addgene N°27077), pCXLE-hSK (addgene N°27078), pCXLE-hUL (addgene N°27080), pEP4 E02S CK2M EN2L (addgene N°20924) and pCXWB-EBNA1 (addgene N°37624). Either the 100 μl tip NEON system or FuGENE6 transfection reagent (Roche, 1814443) was used for the transfection of the plasmids, following the manufacturer´s instructions in both cases. The equine fibroblasts were transfected with different conditions of plasmid concentrations, plasmid combinations and NEON settings, which are detailed in S3 Table. All the conditions were evaluated twice and the EGFP-N1 plasmid was used as control of transfection in each procedure. Once transfected, the cells were cultured in gelatin coated p100 in DMEM 10% SFB medium for 5 days. After that time, they were trypsinized and plated in different dilutions over iMEFs in eqHES medium.