Journal: The FEBS journal

Article Title: Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens.

doi: 10.1111/j.1742-4658.2007.05836.x

Figure Lengend Snippet: Fig. 1. Localization of murine DLAD to lysosomes. (A) Alignment of mouse DLAD and DNase II amino acid sequences. The amino acid sequences of mouse DLAD and DNase II were aligned to give maximum homology by introducing several gaps (–) using GENETYX-MAC genetic information-processing software (version 12) (Genetyx, Tokyo, Japan).The amino acid residues conserved between the two proteins are shown in bold. The amino acids are numbered from the N-terminus, which was determined by Edman degradation of mature rDLAD and rDNase II. The signal sequences are marked by double underlines. Putative active sites carrying the conserved histidine residues (red) are boxed. Potential N-glycosylation sites (Asn-X-Ser ⁄ Thr) are underlined. (B) Production of mouse rDLAD and rDNase II. Mouse rDLAD and rDNase II were produced in human 293T cells transfected with their respective expression plasmid. The DLAD and DNase II secreted into the culture medium were purified, and 0.5 lg of protein was subjected to SDS ⁄ PAGE followed by staining with Coomassie Brilliant Blue. The molecular masses of standard proteins are shown in kDa on the left. (C) Transient expression of rDLAD and rDNase II in 293T cells. Human 293T cells were transfected with the expression vector for Flag-tagged DLAD or DNase II, and cultured for 2 days. The culture sup- ernatants were collected, and the cells were lysed. The Flag-tagged recombinant proteins in the culture supernatants were immunoprecipi- tated with anti-(Flag) protein A–sepharose. Aliquots of the immunoprecipitates (lanes 1 and 3) and cell lysates (lanes 2 and 4) corresponding to 5 · 104 cells were separated by SDS ⁄ PAGE (10%), followed by western blotting with the antibody to Flag. (D) Stable expression of DLAD in HeLa cells. Human HeLa cells were stably transformed with the mouse DLAD expression plasmid. The parental HeLa cells (d, e) or their transformants (a, b, c) were stained with hamster mAb to mouse DLAD (a, d; red) or mouse mAb to human Lamp-1 (b, e; green). In (c), the images obtained with anti-DLAD and anti-Lamp-1 are merged. Scale bar: 20 lm.

Article Snippet: Recombinant DLAD, monoclonal antibody, and analysis of N-terminal amino acid Recombinant (r)DLAD and DNase II were produced by transfecting human 293T cells (ATCC CRL-11268) with pEF-DLAD-Flag or pEF-DNase II-Flag, and the protein secreted into the medium was purified to homogeneity using the anti-Flag M2 affinity gel (Sigma, St Louis, MO).

Techniques: Software, Glycoproteomics, Produced, Transfection, Expressing, Plasmid Preparation, Staining, Cell Culture, Recombinant, Western Blot, Stable Transfection, Transformation Assay

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: The EMBO Journal

Article Title: A novel human fetal lung-derived alveolar organoid model reveals mechanisms of surfactant protein C maturation relevant to interstitial lung disease

doi: 10.1038/s44318-024-00328-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse anti-Mical-L1 , 1:100 , Novus , H00085377.

Techniques: Recombinant, Transduction, CRISPR, Gene Knockout, Expressing, Concentration Assay, Immunofluorescence, Western Blot, Flow Cytometry, Sequencing, Red Blood Cell Lysis, Cell Recovery, Plasmid Preparation, SYBR Green Assay, Reverse Transcription, Software, Microscopy, Magnetic Beads, Transmission Assay