ecotropic booster nature communications  (TaKaRa)

Journal: Nature Communications

Article Title: Non-destructive transcriptomics via vesicular export

doi: 10.1038/s41467-026-72072-w

Figure Lengend Snippet: a Inducible expression of cellular RNA-export machinery enables repeated sampling of vesicle-enclosed transcripts from the supernatant of the same cell population. In contrast, conventional transcriptomics requires cell lysis at each time point. b Schematic of the transfer vector for generating stable NTVE cell lines via ecotropic lentiviral delivery of a modular cassette with a Pgk promoter driving puromycin N -acetyltransferase for selection (~1.1 kbp). A TetON3G transactivator (~0.7 kbp) is coupled via a 2 A peptide, enabling dox-dependent expression of non-infectious Gag mosaics that drive membrane budding. The NTVE cassette (~7 kbp) is flanked by two Long Terminal Repeats (LTR) and stabilized by the Woodchuck Posttranscriptional Response Element (WPRE), a triple helix, and a tRNA. The budding module (~2.1 kbp) can be equipped with different adapters for RNA packaging and affinity handles for purification. A HiBiT tag enables bioluminescence-based quantification of NTVE vesicles. The first two RNA recognition motifs (RRMs, ~0.5 kbp) from poly(A)-binding protein PABPC1 enable the export of polyadenylated mRNAs, while the GCN4 leucine-zipper domain mediates dimerization. c Normalized distribution of hydrodynamic radii of NTVE vesicles after dox induction and enrichment by ultracentrifugation from the supernatant compared to a DMSO vehicle control ( n = 1). d Cryo-electron micrograph of NTVE PABP particles enriched by ultracentrifugation. Scale bar: 50 nm ( n = 1). e Distribution of expression levels for nuclear-encoded genes (grey violin plot) and mitochondrially encoded genes (orange dots) from a stable cell line with inducible expression of NTVE PABP (top) and from transient overexpression of the same construct (bottom). Elevated mitochondrial transcript levels in the supernatant indicate potential membrane perturbation. Reads were filtered for protein-coding genes ( n = 3). f Normalized counts per million (CPM + 10 −3 pseudocounts) from averaged NTVE vesicle triplicates collected in the supernatant of HEK293T cells 72 h after dox induction, plotted against those from the corresponding lysates. Genes encoded on the mitochondrial genome are labeled with their gene symbol and plotted in grey ( n = 3). g Distribution of log10 export ratios in HEK293T cells across detected protein-coding genes from three biological replicates processed identically ( n = 3). The solid line shows the mean density across replicates, and the shaded band indicates the per-bin 95% confidence interval of that mean density (mean ± 1.96 × SEM across replicate histograms). h Quantification of representative mRNA transcripts in the supernatant for NTVE and the NTVE(G2A) PABP control, in which disrupted myristoylation prevents budding ( n = 2, technical replicates). i Accumulation of NTVE particles in the supernatant after dox induction (continuous line: 500 ng/mL; dashed line: 50 ng/mL) monitored by bioluminescence from a HiBiT tag fused to the RRMs of PABPC1. Comparison of two stable cell lines driving the TetON3G transactivator via a Pgk or CAG promoter. Points indicate the mean of three biological replicates with s.d . bounds. j Mean counts per million (CPM) of expressed coding transcripts (CPM > 1) binned by transcript length, exported by NTVE PABP, compared to the corresponding lysates. Input RNA was not enriched for poly(A); total RNA sequencing with rRNA depletion was applied. Data are plotted as the mean of triplicates ± s.d . k Average normalized read depth (top) and number of transcripts (bottom) as a function of nucleotide position relative to the 3′ poly(A) site. Lines represent the mean ± s.d . of triplicates. l Quantification of the number of mRNAs per NTVE particle. NTVE PABP cells were incubated with 500 ng/mL doxycycline for 72 h. Bars represent the mean of biological replicates ± s.d… mRNA was quantified by RNA-seq using spike-in of a known amount of in vitro transcribed mGreenLantern mRNA ( n = 2); particle numbers were quantified by anti-p24 ELISA ( n = 3). Input RNA for ( j , k ) was not enriched for poly(A); total RNA sequencing with rRNA depletion was applied. Source data are provided as a Source Data file. Analysis code is available via Zenodo deposition.

Article Snippet: One day post-seeding, cells were treated with the ecotropic booster according to protocol (Takara Bio, 631471) before the supernatant of one 6-well production was applied to three wells.

Techniques: Expressing, Sampling, Transcriptomics, Lysis, Plasmid Preparation, Selection, Membrane, Purification, Binding Assay, Control, Stable Transfection, Over Expression, Construct, Labeling, Comparison, RNA Sequencing, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Molecular Systems Biology

Article Title: Pooled single-cell screen in colorectal cancer defines transcriptional modules linked to oncogenes

doi: 10.1038/s44320-025-00186-2

Figure Lengend Snippet: ( A ) Design of the transfer plasmid containing the oncogene coding sequence flanked by Gateway cloning sites with a unique 10 bp nucleotide barcode in the 3´-UTR, and a GFP selection marker. ( B ) Experimental workflow of the screen: production of ecotropic lentiviral oncogene library, infection of cell lines with the pooled oncogene library, fluorescence-activated cell sorting of GFP-expressing cells, single-cell analysis using separate libraries for barcodes and transcriptomes. ( C ) Coverage of the oncogenic variants in the screen. Data are cell counts ± SD across the five cell lines. Transgene conditions exceeding 150 cells per replicate and cell line were downsampled, resulting in max. 300 cells for both replicates. ( D ) UMAPs of single-cell transcriptome data per cell line and after integration of replicates, colored by oncogene. Color code as in ( C ). ( E ) Cell line-specific PROGENy pathway activities induced by transgenes. Conditions are labeled by pathway association of the transgenes (see also Fig. ). ( F ) Global transcriptome changes caused by the oncogenes compared to the tdTomato control as quantified by E-Distance. Heatmap shows E-Distance per cell line and replicate. Boxplot shows E-Distance across samples. Box and line show median, Q1 and Q3 values and whiskers extending to 1.5× interquartile range. Rows containing oncogenes are clustered by E-Distance.

Article Snippet: HCT116 cells were pre-treated with Ecotropic Receptor Booster (Takara Bio) prior to viral exposure, according to the manufacturer ́s instructions.

Techniques: Plasmid Preparation, Sequencing, Cloning, Selection, Marker, Infection, Fluorescence, FACS, Expressing, Single-cell Analysis, Single Cell, Labeling, Control