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. 2017 Dec;27(12):2061–2071. doi: 10.1101/gr.222521.117

Figure 1.

Figure 1.

Generation and characterization of Cre-dependent Cas9-expressing pigs. (A) A diagram for TALEN-mediated knock-in of Cre-dependent Cas9-expressing cassette into the pRosa26 locus. Gray triangles, wild-type loxP site; white triangles, mutant loxP2272 site; SA, splice acceptor; TALEN target site and PCR primers (F1, R1, F2, R2, F, and R) are indicated. (B,C) Schematic of two alternative patterns of Cre-mediated activation of SpCas9 and tdTomato: (B, left) Cre recombinase induces inversion of both Neo and iCas9 expression cassettes flanked by two loxP sites, followed by excision of Neo expression cassette flanked by two loxP2272 sites (C); (B, right) Cre recombinase-induced inversion of iCas9 expression cassettes by two loxP2272 sites, followed by excision of Neo expression cassette between two loxP sites (C). After inversion of iCas9 expression cassette and removal of the Neo expression cassette, SpCas9 and tdTomato expression are controlled by the endogenous porcine Rosa26 promoter (C). (D) Morphologically normal piglets were born from SCNT with the pRosa26-iCas9 PFFs. (E) PCR analysis confirmed the correct homologous recombination at the pRosa26 locus in 3/5 cloned piglets. Three positive piglets were all monoallelic modifications, as detected by PCR (F2 + F + R), consistent with those of cells chosen as nuclear donors. Primer pairs are shown in A and in Supplemental Table 3. (F) SpCas9 and tdTomato activations using Cre recombinase in fibroblasts isolated from the ear tissues of cloned piglets shown in D. Cells were infected with Cre-EGFP lentivirus, and the expression of tdTomato and EGFP were observed after 48 h by using a fluorescence microscope. Scale bars, 50 µm. (G) FACS analysis of Cre recombinase-induced tdTomato activation in pRosa26-iCas9 fibroblasts. (H) Western blot analysis was used to directly verify SpCas9 expression in pRosa26-iCas9 fibroblasts infected with lentivirus containing Cre. Cells not infected with Cre lentiviruses and WT cells were used as negative control. (I) H&E staining of the lung, liver, kidney, heart, and spleen of sacrificed wild-type and Cre-dependent Cas9-expressing piglets.