Fig. 1

Generation of the Dox-inducible SpCas9-expressing (DIC) pig line. a Schematic of the gene targeting strategy and SpCas9 expression induced by Dox. The rtTA is contained within the pRosa26 locus. The TRE3G-controlled SpCas9-T2A-tdTomato-expressing cassette is contained within the pHipp11 locus. SpCas9 and tdTomato expression is tightly regulated by Dox. SA, splice acceptor. b Summary of SCNT experiments for generating DIC pigs. c Representative photo of the two DIC founders, 10-month-old 9602-3# (left) and 0101-1# (right). Both are male. d PCR analysis confirmed the correct homologous recombination at the pRosa26 and Hipp11 loci in cloned piglets. e–g Assay of the expression level of SpCas9 and tdTomato protein, induced by Dox, in fibroblasts isolated from the ear tissues of cloned piglets shown in (d). PEFs were treated with or without Dox, and then the Dox-induced tdTomato expression is assayed by FACS analysis (e). Immunofluorescence staining (f) and Western blotting (g) were used to further validate the uniform expression of SpCas9 and tdTomato protein in DIC PEFs with Dox induction. Scale bars, 200 μm