lentiviral transfer plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: CELLISA – a cell-cell binding assay for evaluation of nanovesicle targeting proteins

doi: 10.64898/2026.04.09.717595

Figure Lengend Snippet: (A) Cartoon describing the targets, binder types, and linkers used in this investigation. (B) sCELLISA data identifying candidate binders to Jurkat T cells. Each symbol represents the average of two fields of view from a biological replicate (n = 3), the bar represents the mean, and the error bars represent standard error of the mean. (C) Cartoon depicting the goal of evaluating sCELLISA hits via EV targeting. (D) Quantification of 15k (filled bars) and 120k (outlined bars) EVs targeted to Jurkat T cells with binders identified from the sCELLISA assay. The cellular fluorescence of each condition was background fluorescence subtracted (fluorescence attributable to cells only) and normalized by EV fluorescence determined by NanoFCM. Each bar graph represents an independent experiment, and each bar represents the mean of three biological replicates (n = 3). The error bars represent the standard error of the mean. To evaluate whether binders resulted in increased EV binding compared to the scaffold negative control, a two-way ANOVA was performed, and the Dunnett’s multiple comparison test is shown (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, p > 0.05). (E) Cartoon depicting evaluation of delivery improvement by candidate binders via lentivirus. (F) Quantification of functional titers of unconcentrated lentiviruses expressing mutant VSV-G and the binder indicated on the x-axis. Filled and outlined bars each represent an independent experiment. Each bar represents the mean functional titer as determined via linear regression by calculating the multiplicity of infection using the percent of Jurkat cells expressing dsRedExpress2 (also see Methods: Lentiviral Delivery Studies). Error bars represent the standard error of the slope of the linear regression. To identify if a binder improved delivery compared to the scaffold negative control, a pairwise comparison of each slope from each linear regression to the slope of the scaffold using a one-sample t-test was performed (***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, p > 0.05).

Article Snippet: The lentiviral transfer plasmid was grown in NEB Stable competent E. coli (New England Biolabs C3040) and grown at 30 °C.

Techniques: Fluorescence, Binding Assay, Negative Control, Comparison, Functional Assay, Expressing, Mutagenesis, Infection

Journal: bioRxiv

Article Title: Mechanistic machine learning enables interpretable and generalizable prediction of prime editing outcomes

doi: 10.64898/2026.02.20.706353

Figure Lengend Snippet: (A) Paired pegRNA–target site lentiviral libraries enable high-throughput evaluation of PE2 and PE4 outcomes in HEK293T and HeLa cells. (B) A scatter plot comparing editing efficiencies across Lib-MMR between PE2 (x-axis) and PE4 (y-axis) in HeLa cells. The gray line plots the equation y = x. PE4 consistently outperforms PE2 in HeLa cells. Brighter colors correspond to increased point density. See also Supplementary Figure 2A. (C–H) PE4:PE2 represents the ratio of PE4 editing efficiency to PE2 editing efficiency across library members in Lib-MMR. All PE4:PE2 values are plotted on a logarithmic scale. PE4:PE2 values below 0.25 (HeLa) or 0.5 (HEK293T) are clipped to each respective minimum value. PE4:PE2 values above 1024 (HeLa) or 4 (HEK293T) are clipped to each respective maximum value. (C) Beeswarm plots comparing PE4:PE2 between HeLa cells and HEK293T cells. PE4:PE2 is higher in HeLa cells than in HEK293T cells. (D) A scatter plot comparing PE4:PE2 across library members between cell types. PE4:PE2 correlates well between HEK293T and HeLa cells. Brighter colors correspond to increased point density. (E) Beeswarm plots of PE4:PE2 in HeLa cells for all twelve possible single-base substitutions, sorted by median PE4:PE2. See also Supplementary Figure 2E. (F–H) Beeswarm plots comparing PE4:PE2 in HeLa cells across varying lengths of (F) contiguous substitutions, (G) pure insertions, and (H) pure deletions. See also Supplementary Figure 2F–H. (I) A scatter plot comparing PE4:PE2 editing efficiency ratio of each edit to the ratio of PE2 editing efficiency with and without silent edits across Lib-CV in HeLa cells. Improvements in PE2 efficiency from including silent edits are positively correlated with PE4:PE2 efficiency ratios of the reference edit without any silent edits in HeLa cells. The red line is a total least squares regression line. Brighter colors correspond to increased point density.

Article Snippet: When cells reached 60% confluency (∼16 h after seeding), cells were transfected with 13.3 μg lentiviral transfer plasmid, 6.7 μg pMD2.G (Addgene #12259), and 10 μg psPAX2 (Addgene #12260) with 120 μL Lipofectamine 2000 (ThermoFisher) according to the manufacturer’s protocol.

Techniques: High Throughput Screening Assay