dna plasmids encoding proteins  (GenScript corporation)

Journal: Nature Communications

Article Title: Human cell surface-AAV interactomes identify LRP6 as blood-brain barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

doi: 10.1038/s41467-024-52149-0

Figure Lengend Snippet: a Schematic of AAV cell microarray screen. DNA oligos that encode individual membrane proteins are chemically coupled to slides in a known pattern, reverse transfecting the cells that grow on them and thereby creating spots of cells overexpressing a particular, known protein. Each protein is expressed in duplicate at two different locations on the slide. When AAVs are applied to the slides, enhanced binding can be detected from duplicate cell spots overexpressing cognate AAV receptors. b Known AAV capsid receptor interactions, such as AAVR and LY6A with AAV-PHP.eB, were used to optimize conditions for streptavidin-based detection of biotinylated capsids with two sets of replicate spots. Anti-TGFBR2 antibody was used as a non-AAV positive control. Uncropped blots in source data. c AAVR and LY6A interaction with AAV9.CAP-B22 were used to optimize conditions for anti-AAV9 antibody direct detection of unmodified capsids with two sets of replicate spots. Anti-TGFBR2 antibody was used as a non-AAV control. Uncropped blots in source data. d Pooled AAV capsid screening conditions were optimized by varying the concentrations of individual capsids within the pool to maximize signal to noise after direct detection with anti-AAV9 antibody, with two sets of replicate spots. v.g.: viral genomes. Uncropped blots in source data. e Pooled screening identified preliminary hits which were deconvoluted by individual-capsid screens, identifying previously-unreported potential capsid-binding proteins by direct detection with anti-AAV9 antibody. Transfection control condition detected fluorescent protein reverse transfected along with each receptor. None condition was treated only with anti-AAV9 antibody. Proteins in cyan were identified in all individual AAV screens, and likely represent interactions outside the engineered regions of AAV9. Proteins in magenta specifically bind to at least one engineered capsid. Uncropped blots in source data. Panel a created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: At 80% confluency, cells were transiently transfected with 2.53 µg plasmid DNA encoding a membrane protein hit from the Retrogenix cell microarray screen.

Techniques: Microarray, Membrane, Binding Assay, Positive Control, Control, Transfection

Journal: Pharmaceutics

Article Title: Sucrose Treatment Enhances the Electrotransfer of DNA by Activating Phospholipase A2

doi: 10.3390/pharmaceutics16040475

Figure Lengend Snippet: Effects of phospholipase A2 or V-ATPase inhibition on ET efficiency and cell viability. HCT116 cells were cultured in medium supplemented with or without sucrose (100 mM) for 24 h. Then, the medium was aspirated and replaced with fresh medium containing the indicated concentrations of inhibitors, and the cells were cultured for another 6 h prior to the electrotransfer of DNA encoding enhanced green fluorescence protein (EGFP). Twenty-four hours later, the cells were harvested for flow cytometry analysis to quantify ( A ) the %EGFP+ cells, ( B ) the EGFP expression level, and ( C ) the cell viability. The viability data were normalized according to the mean in the NP control group. The dashed lines in panels ( A , B ) represent the means in the sucrose group. The dashed line in panel ( C ) represents the mean in the NP group. NP, cells were not treated with sucrose or inhibitor, and no ET was performed; NT, ET was performed but the cells were not treated with sucrose or inhibitor. N = 3. #: corrected p -value < 0.05, sucrose group versus NT control; *: corrected p -value < 0.05, group treated with inhibitor and sucrose versus group treated with sucrose alone; &: corrected p -value < 0.05, any group versus NP control; a.u., arbitrary unit.

Article Snippet: DNA plasmid encoding the enhanced green fluorescent protein (EGFP) was purchased from Nova Lifetech Inc. (Hong Kong, China) (pEGFP-N1) and prepared using the PureLink Quick Plasmid Miniprep Kit (ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Cell Culture, Electrotransfer, Fluorescence, Flow Cytometry, Expressing