Journal: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 2. In vitro knockdown of human RHO-T17M expression. (A) Schematic view of construction of 293T stably expressing human RHO protein and transfection of pX601-EFS-SaCas9-U6-sgRNA (SgRNA) plasmid. (B) T7E1 assay indicated that SaCas9/17-Sg1 and SaCas9/17-Sg2 were appeared to cut the mutant sequence specifically, the full-length amplicon was 760 bp, the two truncated amplicons were 510 bp and 250 bp, respectively. (C) The cutting efficacy of two sgRNAs with SaCas9 determined by TA and Sanger sequencing in 293T cells. (D) Rhodopsin expression reduction was determined by WB in RHO17 cells transfected with 17-Sg1 and -Sg2 plasmid, comparing to the RHOwt cells with 17-Sg1 and -Sg2 plasmid. (E) Densitometric analysis of immunoblots performed on RHOwt and RHO17 cells transfected with 17-Sg1 and -Sg2 plasmid, respectively. The experiment was performed in triplicate and presented as mean ± SEM, the significance was calculated using two-tailed paired t-test, ns = not significant, *p<0.05.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: In Vitro, Knockdown, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Sequencing, Amplification, Western Blot, Two Tailed Test

Journal: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 6. Expression of the mutant human RHO allele after gene editing with SaCas9/17-Sg2 in vitro. (A) Schematic view of the different human RHO gene variants created by gene editing. (Top) Map of the pEGFPN1 vector used to overexpress these variants. (Bottom) The description of variants at DNA and protein level. (B) Colocalization of GFP and rhodopsin (4D2, red) in 293T cells transfected with pEGFPN1 vector carrying RHO-WT, RHO-T17M, and four edited RHO-T17M variants, 1 week after transfection. Scale bar = 10 μm. (C) Colocalization of GFP and rhodopsin (4D2, red) in 293T cells transfected with pEGFPN1 vector carrying RHO-5m and four edited RHO-5m variants, 1 week after transfection. Scale bar = 10 μm. (D–F) The number of GFP+ cells and percentage of GFP+ cells expressing rhodopsin per random sight. Nuclei were stained blue by DAPI. Scale bar = 200 μm.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Mutagenesis, In Vitro, Plasmid Preparation, Transfection, Staining

Journal: eLife

Article Title: Allele-specific gene-editing approach for vision loss restoration in RHO-associated retinitis pigmentosa

doi: 10.7554/elife.84065

Figure Lengend Snippet: Figure 10. Examination of SaCas9/17-Sg2 off-target effects in human gDNA using WGS. Identification of SNVs (A) and indels (C) in 293T cells transfected with 17-Sg2 plasmid at the WGS level. The type of SNVs (B) and indels (D) in 293T cells transfected with 17-Sg2 plasmid and untreated cells at the WGS level.

Article Snippet: For the production of lentivirus, 293T cells were transfected with a combination of three plasmids, FUGW- RHO- cDNA or Lenti_SaCRISPR_GFP, Pax2, and vesicular stomatitis virus G protein (VSV- G) plasmid using polyetherimide (PEI) (B600070, ProteinTech Group, Chicago, IL, USA) according to the manufacturer’s protocol.

Techniques: Transfection, Plasmid Preparation

Journal: eLife

Article Title: Interplay between PML NBs and HIRA for H3.3 dynamics following type I interferon stimulus

doi: 10.7554/eLife.80156

Figure Lengend Snippet:

Article Snippet: Human BJ primary foreskin fibroblasts (ATCC, CRL-2522), human IMR90 fetal lung fibroblasts (ATCC, CCL-186), human HEK 293T embryonic kidney cells (Intercell, AG) and mouse MEFs embryonic fibroblasts Pml +/+ or Pml -/- (from Dr. Lallemand-Breitenbach, and whose cell identity was authenticated by STR profiling) were cultivated in DMEM medium (Sigma-Aldrich, D6429) containing 10% of fetal calf serum (FCS) (Sigma-Aldrich, F7524), 1% of penicillin/streptomycin (Sigma-Aldrich, P4458), at 37 °C under 5% CO2 and humid atmosphere.

Techniques: Transduction, Recombinant, Plasmid Preparation, Clone Assay, Mutagenesis, Sequencing, Quantitative RT-PCR, Concentration Assay, In Situ, Software, ChIP-sequencing