Journal: bioRxiv

Article Title: Circular single-stranded DNA is a superior homology-directed repair donor template for efficient genome engineering

doi: 10.1101/2022.12.01.518578

Figure Lengend Snippet: A . Schematic diagram of the GFP reporter cssDNA targeting RAB11A locus and TRAC locus (Upper). GFP reporter knock-in efficiency with cssDNA donor templates in human primary T cells. Immediately after electroporation, cells were treated with either DMSO or 1 µM M-3814 for 24 hours. The knock-in efficiency was examined on Day 7 post electroporation. B . Secreted IFN-γ and TNF-α cytokines levels in cultured primary T medium after electroporation with buffer, dsDNA, cssDNA or mRNA molecules at 1 µg/million cells. Conditioned media were collected for cytokine analysis using the Ella immunoassay platform with selected panels. C . Gene set enrichment analysis (GSEA) analysis of interferon gamma response genes on the differential expression genes (DEG) of RNA samples from dsDNA or cssDNA treated primary T cells. NES: normalized enrichment score; FDR q-value: False Discovery Rate-adjusted p-value. **** p<0.0001 One-Way ANOVA Bonferroni post hoc test between indicated groups.

Article Snippet: T cells were activated for 2 days with T Cell TransAct (Miltenyi) before electroporation.

Techniques: Knock-In, Electroporation, Cell Culture, Quantitative Proteomics

Journal: bioRxiv

Article Title: Circular single-stranded DNA is a superior homology-directed repair donor template for efficient genome engineering

doi: 10.1101/2022.12.01.518578

Figure Lengend Snippet: A . Schematic diagram of a nonviral CAR-T cell engineering process. Pan T cells were isolated from peripheral blood and activated on day 0 with anti-CD3/anti-CD28 TransAct. Cells were electroporated using the Lonza nucleofector on day 2 with Cas9 RNPs + cssDNA HDR donor templates and then expanded for a total of 7–10 days. B . Representative Day 7 flow cytometry graphs showing CAR knock-in for Mock (un-engineered) and cssDNA-engineered T cells with DMSO or 1 µM M-3814 treatments. C . Representative Day 7 flow plots and quantifications of CAR knock-in for mock (un-engineered), cssDNA- or AAV6-engineered T cells. Cells were treated with 1 µM M-3814 for 1 day immediately after electroporation. D . Expansion ability of engineered CAR-T cells. E . In vitro killing of NALM6 acute lymphoblastic leukemia cell line with cssDNA-, AAV6-engineered CAR-T cells in comparison to unmodified T cells from the same blood donor after 24 hours of co-culture. The in vitro killing measured by live cell imaging using the IncuCyte® live cell imaging system. F . The growth curve of target NALM6 cells when co-cultured with un-engineered, cssDNA- or AAV6-engineered CAR-T cells at various effector to target (E:T) ratios.

Article Snippet: T cells were activated for 2 days with T Cell TransAct (Miltenyi) before electroporation.

Techniques: Isolation, Flow Cytometry, Knock-In, Electroporation, In Vitro, Comparison, Co-Culture Assay, Live Cell Imaging, Cell Culture

Journal: Cell stem cell

Article Title: Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma.

doi: 10.1016/j.stem.2017.04.009

Figure Lengend Snippet: Figure 7. a-ITGA7 Impairs GBM Tumor Growth and Invasion In Vivo (A) Luciferase activity detected with a Xenogen IVIS 100 small animal in vivo imaging system, 5 days post-sc transplantation of 5 X 105 GSC1-LUC cells in mice either treated with anti-ITGA7 (a-ITGA7) antibody or isotype control (i.p.). (B) Analysis of sc GSC1-LUC tumor growth over time in mice treated either with isotype control or with a-ITGA7 antibody (1.4A12). Shown is the average photon count and SEM of n = 6 mice/group measured by Xenogen IVIS 100small animal in vivo imaging system (***p< 0.001, two-tailed Student’s t test). See also FigureS7. (C) Analyses of the tumor volumes 5 months after ceasing the anti-ITGA7 treatment. Shown is the size of the sc tumors of n = 6 mice/group measured by calliper (**p < 0.01, two-tailed Student’s t test).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER PE Mouse monoclonal anti EphA2 (clone 371805) R&D Cat#FAB3035P; RRID:AB_11128496 APC Mouse monoclonal anti CD44 BD Biosciences Cat# 560890 Mouse monoclonal anti CD44 (clone DF1485) DAKO Cat#M7082; RRID:AB_2076596 Mouse monoclonal anti ITGA7 Miltenyi Bioteck Cat#130-103-774 Mouse monoclonal anti murine ITGA7 (clone 6A11) Acris Cat#AM20012AF-N; Please See Table S3 for Reverse Phase Protein Microarray Antibodies used N/A N/A Biological Samples GSC (Glioblastoma Stem Cells) (Marziali et al., 2016; Ricci-Vitiani et al., 2008) N/A Chemicals, Peptides, and Recombinant Proteins EGF Peprotech Cat#100-15 bFGF Peprotech Cat#100-18B Apotransferrin Sigma Cat#T2252 Putrescine Sigma Cat#P5780 Sodium Selenite Sigma Cat#S5261 Bovine Serum Albumin Sigma Cat#A2153 Progesterone Sigma Cat#P8783 Insulin Sigma Cat#I3536 HAT Supplement 50X Thermo Scientific Cat#21060017 HT Supplement 100X Thermo Scientific Cat#11067030 BM Condimed H1 Hybridoma Suppl.

Techniques: In Vivo, Luciferase, Activity Assay, In Vivo Imaging, Transplantation Assay, Control, Two Tailed Test