Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: In Vivo Intra-Uterine Delivery of TAT-Fused Cre Recombinase and CRISPR/Cas9 Editing System in Mice Unveil Histopathology of Pten/p53-Deficient Endometrial Cancers.

doi: 10.1002/advs.202303134

Figure Lengend Snippet: Figure 2. Recombinant TAT-Cre exhibits recombinase activity on endometrial epithelial cells in vitro and in vivo. A) Representative images and quantifi- cation of tdTomato positive (mT) or GFP positive (mG) monolayer endometrial epithelial cells isolated from mT/mGf/f mouse ears 4 days after being treated with the following TAT-CRE concentrations: 0 (0), 17.6 (D1), 35.2 (D2), 88 (D3), 176 (D4), and 352 μg mL−1 (D5). Data come from n = 3 indepen- dent experiments. *p<0.5 ****p<0.0001, using a one-way ANOVA analysis, followed by a Bonferroni multiple comparison test. B) Representative images and quantification of tdTomato positive (mT) or GFP positive (mG) organoids of endometrial epithelial cells isolated from mT/mGf/f mouse ears 4 days after being treated with the following TAT-Cre concentrations: 0 (0), 17.6 (D1), 35.2 (D2), 88 (D3), 176 (D4), and 352 μg mL−1 (D5). Data come from n = 3 independent experiments. *p<0.5 ****p<0.0001, using a one-way ANOVA analysis, followed by a Bonferroni multiple comparison test. C) Diagram depicting intrauterine administration of 88 μg of recombinant TAT-Cre per uterine horn of mT/mGf/f mouse. D) Right, representative images images obtained from red (tdTomato) and green (mGFP) fluorescence at 4X of mT/mGf/f endometria 4 months after intravaginal administration of 88 μg of recombinant TAT-Cre. Scale bar at 2 mm. Left, 20x magnification of the indicated framed areas where the membranous mGFP labeling is appreciated in endometrial cells 4 month after TAT-Cre-mediated gene ablation. E) Representative images of GFP immunohistochemistry on endometrial tissue sections from mT/mGf/f endometria 4 months after intravaginal administration of 88 μg of recombinant TAT-Cre showing areas of high recombination (left image) or low recombination (right image). Table shows the average of the percent of GFP positive (GFP+) on epithelial and stromal compartment. Cells were scored from at least 10 sections from 3 different areas from 3 independent mice.

Article Snippet: Cloning, Expression, and Isolation of Recombinant TAT-CRE: Cre recombinase DNA was amplified by PCR using pCAG-Cre (Addgene #13 775) as nloaded from https://onlinelibrary.w iley.com /doi/10.1002/advs.202303134 by C A PE S, W iley O nline L ibrary on [11/01/2024].

Techniques: Recombinant, Activity Assay, In Vitro, In Vivo, Isolation, Comparison, Labeling, Immunohistochemistry