Journal: Nature biomedical engineering

Article Title: Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose.

doi: 10.1038/s41551-018-0293-z

Figure Lengend Snippet: Fig. 1 | Expression of engineered hBChE via genome editing in skin epidermal stem cells. a, Targeting strategy for the expression of engineered hBChE. The targeting vector contains two Rosa26 homology arms, which flank the expression cassette for hBChE and a selection marker (the puromycin-resistant gene Puro) driven by the constitutive promoter ubiquitin C promoter (UbiC). hBChE and Puro are separated by a self-cleavable peptide, T2A. b, Integration of the targeting vector into the Rosa26 locus was verified by PCR (left) and Southern blotting (right). Positive clones with hBChE integration (BChE-knock-in (KI)) display an additional band of the expected size. Three independent experiments were performed with similar results. bp, base pairs; kb, kilobases. c, Confirmation of hBChE expression in targeted cells by immunoblots with different antibodies. Numbers to the left indicate molecular weight markers. kD, kilodalton. Full scans of the western blots are provided in Supplementary Fig. 1d. Three independent experiments were performed with similar results. d, Confirmation of the secretion of engineered hBChE in the culture media by ELISA. The box plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentiles (whiskers), 1st and 99th percentiles (solid triangles), and minimum and maximum measurements (solid squares). e, Cocaine hydrolysis activity in vitro. Cell culture supernatants were collected from cells targeted by hBChE or mouse BChE (mBChE). Cocaine hydrolysis activity was examined by a clearance assay in vitro. n = 3 independent experiments. Error bars represent s.d. and the central measure represents the mean. f, Cell cycle profiles of control and hBChE-expressing epidermal stem cells. Propidium iodide (PI) was used to identify the proportions of cells that are in various stages of the cell-cycle through flow cytometry and measures of relative DNA content. Three independent experiments were performed with similar results.

Article Snippet: Rabbit polyclonal BChE antibody was obtained from Proteintech.

Techniques: Expressing, Plasmid Preparation, Selection, Marker, Ubiquitin Proteomics, Southern Blot, Clone Assay, Knock-In, Western Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay, Activity Assay, In Vitro, Cell Culture, Control, Flow Cytometry

Journal: Nature biomedical engineering

Article Title: Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose.

doi: 10.1038/s41551-018-0293-z

Figure Lengend Snippet: Fig. 4 | Expression of hBChE in human epidermal stem cells with CRISPR. a, AAVS1-targeting strategy for the expression of engineered hBChE. The targeting vector contains two AAVS1 homology arms, which flank the expression cassette for hBChE and a selection marker (the puromycin-resistant gene Puro) driven by the constitutive promoter ubiquitin C promoter (UbiC). hBChE and Puro are separated by a self-cleavable peptide, T2A. b, Integration of the targeting vector into the AAVS1 locus was verified by Southern blotting. Positive clones with hBChE integration (BChE-KI) display an additional band of the expected size. Three independent experiments were performed with similar results. kb, kilobases. c, The expression of hBChE was confirmed in targeted cells by immunoblots with different antibodies as indicated. Full scans of the western blots are available in Supplementary Fig. 3e. Three independent experiments were performed with similar results. d, Secretion of engineered hBChE in the culture media was confirmed by ELISA (n = 3 independent experiments). Box plots were generated with the same elements as described in Fig. 1d. e, Nude mice were grafted with organotypic human skin culture. Intravital imaging shows the efficient incorporation of grafted cells expressing luciferase (right) or control cells (left) on engraftment. f, Sections of grafted skin were immunostained with different antibodies (keratin 10 (Krt10), a marker for early epidermal differentiation; loricrin (Lor), a marker for late epidermal differentiation; and β4-integrin (CD104) (β4), a marker for skin basement membrane) as indicated. Dashed lines denote the basement of the skin. Scale bar, 50 μm. DAPI, 4‘,6-diamidine-2’-phenylindole dihydrochloride; Der, dermis; Epi, epidermis. g, Mice were grafted with control or hBChE skin organoids. The presence of hBChE in the blood was determined by ELISA for 8 wk after engraftment (n = 3 mice per group).

Article Snippet: Rabbit polyclonal BChE antibody was obtained from Proteintech.

Techniques: Expressing, CRISPR, Plasmid Preparation, Selection, Marker, Ubiquitin Proteomics, Southern Blot, Clone Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Imaging, Luciferase, Control, Membrane