Journal: Journal of Extracellular Vesicles

Article Title: Distinguishing Pseudotransduction and True Transduction Enables Characterization and Bioengineering of Extracellular Vesicle‐Adeno‐Associated Virus Vectors

doi: 10.1002/jev2.70258

Figure Lengend Snippet: A Cre recombinase‐based dual reporter system distinguishes mixed delivery and transduction. (A) Schematic depicting the expected function of the stoplight dual‐reporter system in which Cre expressed in recipient cells acts upon incoming AAV genomes to switch them from expression of a red reporter to a green reporter. Free AAV mediates transduction only (green); EV‐AAV mediates both mixed delivery (red) and transduction (green) and sometimes both (yellow); No Rep/Cap EVs can only mediate mixed delivery (red). (B) Cre‐responsive plasmids were evaluated in this experiment. (C, D) Mixed delivery (C) and transduction (D) conferred by various vector compositions. Note: some mCherry gene expression could occur in recipient cells prior to Cre‐mediated recombination (or in the possible absence of recombination), such that (C) includes both mixed delivery and this ambiguous de novo gene expression. Samples were normalized to include 1e9 vector genomes per well (for AAV crude lysate conditions) or a volume‐equivalent of the AAV2 crude lysate condition for No Rep/Cap crude lysate conditions (10 4 recipient cells). Experiments were performed in biological triplicate. One of two independent experiments is shown (second experiment: Figure S10 ). Error bars indicate the standard error of the mean. Multicomparison statistical analysis was performed using a two‐way ANOVA test, followed by Tukey's multiple comparison test to evaluate specific comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). NLS, nuclear localization sequence; ns, not significant; PBS, phosphate‐buffered saline.

Article Snippet: RepCap plasmids for AAV2 (pRep2Cap2) and AAV6 (pRep2Cap6) were gifts from the Vector Core at the University of Pennsylvania (Penn Vector Core (RRID: SCR_022432)). pcDNA is plasmid pPD005 (Addgene plasmid # 138749; http://n2t.net/addgene:138749 ; RRID:Addgene_138749) (Donahue et al. ). psPAX2 and pMD2.G plasmids were gifted by William Miller from Northwestern University. pHIE822 was created by introducing point mutations (K47Q, R354A) in the VSV‐G‐encoding gene of pMD2.G using site‐directed mutagenesis by PCR. pCMV‐VSV‐G(P127D)‐Myc (pJB042) was a gift from Wesley Sundquist (Addgene plasmid # 80055; http://n2t.net/addgene:80055 ; RRID:Addgene_80055) (Votteler et al. ). pHIE963 was created using standard restriction enzyme cloning to insert a Cre recombinase gene into a third‐generation lentiviral transfer vector provided by Twist Bioscience (pTwist Lenti SFFV PuroR).

Techniques: Transduction, Expressing, Plasmid Preparation, Gene Expression, Comparison, Sequencing, Saline

Journal: bioRxiv

Article Title: SFPQ-TFE3 gene fusion reciprocally regulates mTORC1 activity and induces lineage plasticity in a novel mouse model of renal tumorigenesis

doi: 10.1101/2024.11.21.624702

Figure Lengend Snippet: (A) Dual IHC for PAX8 (brown) and GPNMB (teal) in tamoxifen-injected, SFPQ-TFE3 LSL ; Pax8-CreERT transgenic mice and age-matched, littermate controls, at 2 weeks following injection of tamoxifen. Scale bar = 100 µm. (B) H&E (top row) and dual PAX8/GPNMB IHC staining (bottom row) of kidneys from representative tamoxifen-injected, SFPQ-TFE3 LSL ; Pax8-CreERT transgenic mice and age-matched, littermate controls, at 3.5 months following injection of tamoxifen. Scale bar= 100 µm. (C) H&E (top row), dual PAX8/GPNMB IHC staining (middle row) and PAX2 IHC staining (bottom row) of kidneys from representative age-matched, control and SFPQ-TFE3 LSL ; Ksp-Cre transgenic mice at post-natal day 15. Scale bar = 100 µm. (D) H&E (top row), dual PAX8/GPNMB IHC staining (middle row) and PAX2 IHC staining (bottom row) of kidneys from representative age-matched, control and PRCC-TFE3 LSL ; Ksp-Cre transgenic mice at 7 to 9 months. (E) Digital quantification of mean nuclear PAX8 H-scores in GPNMB-cells (blue) and GPNMB+ cells (red) at day 1 and day 15 (Fix Figure), from experiments in C , as depicted by scatter plots, with the central line denoting the median. The number of biological replicates analyzed for both, GPNMB – and + cells were n=11 (day 1) and n=9 (day 15). Statistical analyses were performed using two-tailed Mann-Whitney test. (F) Digital quantification of mean nuclear PAX8 H-scores in GPNMB-cells (blue) and GPNMB+ cells (red) at 4 months and 7 months, from experiments in D , as depicted by scatter plots, with the central line denoting the median. The number of biological replicates analyzed for both, GPNMB – and + cells were n=9 at 4 and 7 months. Statistical analyses were performed using two-tailed Mann-Whitney test. (G) Gene Set Enrichment Analysis (GSEA) comparing 15-day STK (top panels), 3.5-month tamoxifen-treated, STP (middle panels) and 7-month PTK (bottom panels), transgenic kidneys and their controls, for genes differentially expressed in renal TSC-related PEComas from . (H) Immunoblotting of lysates from primary renal tubular epithelial cells from SFPQ-TFE3 LSL transgenic mice treated with control or Cre-recombinase expressing adenovirus in vitro for the indicated markers. Source data are provided as a Source data file.

Article Snippet: 2) Mice hemizygous for the Ksp -Cre recombinase knockin gene (Strain Number: 012237) (The Jackson Laboratory).

Techniques: Injection, Transgenic Assay, Immunohistochemistry, Control, Two Tailed Test, MANN-WHITNEY, Western Blot, Expressing, In Vitro