tmprss2 plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: The proton-activated chloride channel inhibits SARS-CoV-2 spike protein-mediated viral entry through the endosomal pathway

doi: 10.1101/2025.03.12.642872

Figure Lengend Snippet: A) Western blot validating the inducible expression of PAC and stable expression of ACE2 and TMPRSS2. Representative image of three experiments. B) SARS-CoV-2 spike-mediated pseudoviral entry for HEK 292T ACE2 cells with or without PAC and TMPRSS2 expression. % Relative Luciferase Units (RLU) were normalized to the mean of the controls that did not express PAC or TMPRSS2. n = 15 wells from 5 experiments, unpaired two-tailed student’s t- test with Welch’s correction. Bars represent mean ± SEM; **** p<0.0001 .

Article Snippet: 6 μg of ACE2 plasmid (pLENTI_ACE2_PURO, Addgene 155295), TMPRSS2 plasmid (pLEX307-TMPRSS2-blast, Addgene158458) or hPAC-mCherry were co-transfected with 2 μg of packaging plasmids [pVSV-G (pMD2.G, Addgene 12259), pMDL (pMDLg/pRRE, Addgene 12251) and pRSV (pRSV-REV, Addgene 12253)].

Techniques: Western Blot, Expressing, Luciferase, Two Tailed Test

Journal: eLife

Article Title: Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

doi: 10.7554/eLife.89035

Figure Lengend Snippet: ( A ) Schematic representation of the workflow to generate clonal virus populations. Right after transfection, cells were diluted to less than 0.5 virus-producing cells/well in 96-well plates. Then, 7 d post-transfection, the supernatant was transferred onto Vero E6-TMPRSS2 cells. Plates were observed until a cytopathic effect (CPE) became apparent. ( B ) Clonal virus populations arising from a single virus-producing cell were identified after supernatant transfer onto Vero E6-TMPRSS2 cells. CPE of infectious virus was assessed by microscopy and virus was collected for further analysis. Thereafter, plates were fixed and stained for the fast enumeration of positive wells (in light blue).

Article Snippet: Vero E6-TMPRSS2 cells were generated by transduction with a second-generation lentiviral vector pLEX307-TMPRSS2-blast (Addgene plasmid #158458) and selected for 2 wk in DMEM containing 20 µg/mL of Blasticidin (Cat# SBR00022, Sigma-Aldrich).

Techniques: Virus, Transfection, Microscopy, Staining

Journal: eLife

Article Title: Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

doi: 10.7554/eLife.89035

Figure Lengend Snippet:

Article Snippet: Vero E6-TMPRSS2 cells were generated by transduction with a second-generation lentiviral vector pLEX307-TMPRSS2-blast (Addgene plasmid #158458) and selected for 2 wk in DMEM containing 20 µg/mL of Blasticidin (Cat# SBR00022, Sigma-Aldrich).

Techniques: Variant Assay, Virus, Recombinant, Plasmid Preparation, Expressing, Sequencing, Nucleic Acid Purification, Software, Microscopy, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

doi: 10.7554/eLife.89035

Figure Lengend Snippet: The most commonly used reverse genetics systems for the rescue of recombinant SARS-CoV-2 are listed and the prominent intermediate steps are depicted. Note that this is a schematic summary and additional steps (such as purification, linearization before transcription, etc.) or small aberrations of the protocol (e.g., different starting material) can apply. The first groups reporting the successful adaptation to SARS-CoV-2 are mentioned. For the CLEVER method, additionally the direct mutagenesis within the initial RT-PCR step is depicted (mutated sites marked with red asterisks). Repeated icons are only labeled once. DNA fragments are represented as blue lines. T7, T7 RNA polymerase; BAC, bacterial artificial chromosome; YAC, yeast artificial chromosome; CPER, circular polymerase extension reaction; ISA, infectious subgenomic amplicons; CLEVER, CLoning-free and Exchangeable system for Virus Engineering and Rescue.

Article Snippet: Recombinant DNA reagent , pLEX307-TMPRSS2-blast (plasmid) , Addgene , Cat# 158458 , –.

Techniques: Recombinant, Purification, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Labeling, Cloning, Virus

Journal: eLife

Article Title: Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

doi: 10.7554/eLife.89035

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pLEX307-TMPRSS2-blast (plasmid) , Addgene , Cat# 158458 , –.

Techniques: Variant Assay, Virus, Recombinant, Plasmid Preparation, Expressing, Sequencing, Nucleic Acid Purification, Clinical Proteomics, One Step RT-PCR, Software, Microscopy, Real-time Polymerase Chain Reaction

Journal: Virology

Article Title: An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E.

doi: 10.1016/j.virol.2023.109889

Figure Lengend Snippet: Fig. 1. Expression of CD13 and TMPRSS2 in engineered A549 cells. 30 μg of protein from cell lysates was loaded onto a 4%–12% Bis-Tris gel and sepa rated by SDS-PAGE before expressions of (A) CD13 and (B) TMPRSS2 were analyzed by Western Blot. GAPDH detection is included as the loading control. WT A549 cells are included as a negative control, whereas HAE lysates are included as a positive control for TMPRSS2.

Article Snippet: The mix consisted of 27 μg of PEI (Sigma-Aldrich), 9 μg of a vector carrying either the CD13/aminopeptidase N sequence (pLEX307-APN-G418 was a gift from Alejandro Chavez & Sho Iketani; Addgene plasmid #158456) or the TMPRSS2 sequence (pLEX307-TMPRSS2-blast was a gift from Alejandro Chavez & Sho Iketani; Addgene plasmid #158458).

Techniques: Expressing, SDS Page, Western Blot, Control, Negative Control, Positive Control