Fig. 2: Direct protein delivery of Cre recombinase mediated by cell-penetrating peptides.
a, Schematic cartoon of colour-switch Cre-reporter cell line. GFP is constitutively expressed, while a stop codon prevents expression of RFP. Upon Cre-mediated excision of GFP and the stop codon, RFP expression replaces expression of GFP. b, Various formulations of cell-penetrating Cre mediate excision of the GFP gene and induce expression of RFP measured by fluorescence microscopy (top) and flow cytometry (bottom). From left to right: purified Cre recombinase delivered alone; with Lipofectamine 3000 (L3000); with fused CPP5 covalent cell-penetrating peptide; with fused TAT; with fused ANTP; with 6×His-CM18-PTD4 non-covalent cell-penetrating peptide (6H-CM18-PTD4); with fused ANTP cell-penetrating peptide and 6×His-CM18-PTD4 peptide. Scale bar, 200 μm. c, Quantification of Cre-mediated GFP to RFP conversion as a function of direct protein delivery with Cre recombinase alone or with fused cell-penetrating peptides, as measured by flow cytometry. Three biological replicates with 2 analytical replicates each, mean ± s.d. 1 µM corresponds to 41.9 µg ml−1. d, Quantification of enhancement of Cre-mediated GFP to RFP conversion with increasing concentrations of 6×His-CM18-PTD4 peptide at a protein concentration of 0.5 µM. Symbols as in c. Three biological replicates with 2 analytical replicates each, mean ± s.d. e, Schematic cartoon of genetic construct of ROSA mT/mG Cre-reporter mouse model. In the mice, tdTomato is constitutively expressed, while a stop signal prevents expression of eGFP. Upon Cre-mediated excision of tdTomato and the stop signal, eGFP expression replaces expression of tdTomato. f, Protein delivery to skin fibroblasts isolated from the ROSA mT/mG Cre-reporter mice, as observed with a fluorescence microscope (top) and quantified by flow cytometry (bottom), 72 h post delivery. Scale bar, 100 μm. g, Quantification by flow cytometry of Cre recombinase delivery into ROSA mT/mG Cre-reporter mouse primary fibroblasts. Two separate experiments with 2 analytical replicates each, mean ± s.d. h, Schematic cartoon representing retinal cross-section orientation. RPE, retinal pigment epithelium; PR OS, photoreceptor outer segment; PR IS, photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. Created in part with BioRender.com: i66o107. i–p, Cre-mediated tdTomato to eGFP expression, 1 week after subretinal delivery of Cre recombinase measured by two-photon excitation microscopy. The RPE layer is orientated towards the top, denoted with open yellow triangles. Scale is provided in micrometres. i, AAV2/1-CMV-Cre; j, VSV-G pseudotyped Cre eVLP; k, Cre recombinase alone; l, Cre recombinase delivered with Lipofectamine 3000; m, Cre recombinase delivered with 6×His-CM18-PTD4 peptide; n, CPP5-fused Cre recombinase; o, TAT-fused Cre recombinase; p, ANTP-fused Cre recombinase. 3D video reconstructions of i–p are available as Supplementary Videos 1–8.
