Figure 3.

Delivery of active Cre recombinase into mammalian cells in vitro and in vivo. a) Cre-mediated recombination in HeLa cells transiently transfected with pCALNL-DsRed2 and treated with +36 GFP-Cre, Tat-Cre, Arg₁₀-Cre, or penetratin-Cre for 4 h at 37 °C. The image is an overlay of DsRed2 signal and brightfield images of HeLa cells transfected with pCALNL-DsRed2 and treated with 100 nM +36 GFP-Cre. b) Cre-mediated recombination in 3T3.LNL.LacZ cells treated with +36 GFP-Cre, Tat-Cre, Arg₁₀-Cre, or penetratin-Cre for 4 h at 37 °C. The image is of 3T3.loxP.lacZ cells treated with 500 nM +36 GFP-Cre and stained with X-Gal. c) Cre-mediated recombination in BSR.LNL.tdTomato cells treated with +36 GFP-Cre, Tat-Cre, Arg₁₀-Cre, or penetratin-Cre for 4 h at 37 °C. The image is an overlay of tdTomato signal and brightfield images of BSR.LNL.tdTomato cells treated with 100 nM +36 GFP-Cre. d) Identical to panel c but with the addition of 100 μM chloroquine during and after protein treatment. In panels a−d, error bars reflect the standard error of three independent biological replicates. e) Fluorescence microscopy of a retinal section of a CD1 adult mouse injected with 0.5 μL of 100 μM +36 GFP. The retina was harvested and analyzed 6 h after injection. GFP fluorescence is shown in green, and DAPI nuclear stain is shown in blue. f) Retinal sections of neonatal RC::PFwe mouse pups harboring a nuclear LacZ reporter of Cre activity. Three days after injection of 0.5 μL of 40 μM wild-type Cre, Tat-Cre, or +36 GFP-Cre, retinae were harvested, fixed, and stained with X-gal. Dots on the graph represent the total number of recombined cells counted in each retina. The horizontal bar represents the average number of recombined cells per retina for each protein injected (n = 4 for wild-type Cre, n = 6 for Tat-Cre, n = 6 for +36 GFP-Cre).