Fig. 1

CRISPR/dCas9 activation (CRISPRa) system mediated overexpression of IL10 in Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs). a Immunophenotypic profile of BM-MSCs. Flow cytometry histograms after three passages show the expression of selected surface molecules (including CD29, CD44, CD90, CD34 and CD45). Cells were stained with FITC-labeled antibodies or isotype control antibody. The BM-MSCs were positive for CD29, CD44, and CD90 but negative for CD34 and CD45. b Establishment of streptozotocin-induced diabetic model. 6–8-week-old BALB/c mice were intraperitoneal (i.p.) injection with Streptozotocin (STZ) (40 mg/kg) for 5 consecutive days. The IL-10 expressions in mRNA level (c) and protein level (d) of BM-MSCs isolated from healthy mice and streptozotocin (STZ)-induced diabetic mice was examined by real-time PCR (RT-PCR) and Western blotting, respectively. Data represent means ± SD. ***p < 0.001, n = 3. e Production of IL-10 by BM-MSCs and diabetic BM-MSCs. The IL-10 levels in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Data represent means ± SD. **p < 0.005, n = 3. f Schematic diagram of CRISPR/dCas9 activation plasmids. The engineered plasmid dCas9-VP64-MS2 gRNA encodes Cas9 that lacked nuclease activity (dCas9) and gRNA, the plasmid MS2-p65-HSF1 encodes MS2-P65-HSF1 transcriptional activation complex. The gRNA Sequence was designed by the online CRISPR Design Tool (http://tools.genome-engineering.org). BM-MSCs isolated from diabetic mice were transfected with CRISPRa plasmids expressing IL10 (pCas9/gIL10) or control plasmids (pCas9/gCtrl). The IL10 expression (g) and IL10 production (h) in diabetic BM-MSCs were examined by Western blotting and ELISA 48 h post transfection. Data represent means ± SD. ***p < 0.001, n = 3