enhanced green fluorescent protein (egfp) mrna  (TriLink)

Journal: bioRxiv

Article Title: Buffer specificity of ionizable lipid nanoparticle transfection efficiency and bulk phase transition

doi: 10.1101/2025.01.17.633509

Figure Lengend Snippet: eGFP-mRNA LNPs encapsulation efficiency percentage in buffer citrate, phosphate and acetate 50 mM at pH 3. Encapsulation efficiencies are namely citrate 94 ± 4, phosphate 79 ± 8 and acetate 97 ± 2.

Article Snippet: ARCA eGFP (Enhanced Green Fluorescent Protein) mRNA (APExBIO) was encapsulated in an LNP of four lipid components (DLin-MC3-DMA, DSPC, Cholesterol, DMPE-PEG2000) in a 50:10:38.5:1.5 molar ratio. mRNA and buffer (pH 3) were prepared in aqueous solution (0.075 mg/mL) while the lipid components (1.11 mg/mL MC3, 0.27 mg/mL DSPC, 0.52 mg/mL cholesterol, 0.15 mg/mL DSPE-PEG2k) were dissolved in ethanol.

Techniques: Encapsulation

Journal: bioRxiv

Article Title: Buffer specificity of ionizable lipid nanoparticle transfection efficiency and bulk phase transition

doi: 10.1101/2025.01.17.633509

Figure Lengend Snippet: Single cell transfection experiments: (A) Culturing cells on microfabricated single-cell arrays allows recording of hundreds of single-cell fluorescence trajectories in parallel. (B) Single cell expression time courses of eGFP-mRNA/LNPs (gray lines) show different fluorescence kinetics for different preparation buffers (averaged time courses shown in color). (C) Scatter plot of single cell expression efficiencies (area under the curve) versus expression onset times (D) Mean values of single cell efficiencies (C) for the three LNP buffer conditions exhibit an apparent Hofmeister ordering. Data for lipofectamine transfection are shown as reference. In general, faster onset is correlated with higher protein expression. Error bars indicate standard error of the mean.

Article Snippet: ARCA eGFP (Enhanced Green Fluorescent Protein) mRNA (APExBIO) was encapsulated in an LNP of four lipid components (DLin-MC3-DMA, DSPC, Cholesterol, DMPE-PEG2000) in a 50:10:38.5:1.5 molar ratio. mRNA and buffer (pH 3) were prepared in aqueous solution (0.075 mg/mL) while the lipid components (1.11 mg/mL MC3, 0.27 mg/mL DSPC, 0.52 mg/mL cholesterol, 0.15 mg/mL DSPE-PEG2k) were dissolved in ethanol.

Techniques: Transfection, Fluorescence, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Production of site-specific antibody conjugates using metabolic glycoengineering and novel Fc glycovariants

doi: 10.1016/j.jbc.2024.108005

Figure Lengend Snippet: Fc glycovariant conjugation to biomaterials enables targeted gene delivery. A , schematic of detection scheme for flow cytometry analysis of azido-modified trastuzumab glycovariants linked to DBCO-coated magnetic microparticles. Antibody conjugation is detected with a fluorescent anti-Fab antibody and target antigen binding is detected using biotinylated HER2 and secondary fluorescent streptavidin staining. Representative flow cytometry plots are shown below. B , cartoon depicting poly(beta-amino ester) (PBAE) nanoparticle encapsulating cyanine-5 (Cy5)-labeled enhanced green fluorescent protein (eGFP)-encoding mRNA conjugated to trastuzumab glycovariant antibodies. C and D , nanoparticle uptake in transfected HER2 + SKBR3 ( C ) and HER2 - MDA-MB-231 ( D ) cells, normalized to the unconjugated control for each concentration. E and F , eGFP expression in transfected SKBR3 ( E ) and MDA-MB-231 ( F ) cells, normalized to the unconjugated control for each concentration. Statistical significance was determined by two-way ANOVA with a Dunnett post hoc test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001 (n = 4).

Article Snippet: To measure both uptake and gene expression simultaneously, enhanced green fluorescent protein (eGFP) mRNA (CleanCap, 5-methoxyuridine-modified, Trilink Biotchnologies) was labeled with Cy5 using the Label IT Nucleic Acid Labeling Kit (Mirus Bio) according to the manufacturer's instructions.

Techniques: Conjugation Assay, Flow Cytometry, Modification, Binding Assay, Staining, Labeling, Transfection, Control, Concentration Assay, Expressing