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VV(Li)-RucGFP-mTyr-GusA

  • 0.25 mL / Standard (OV4002) $ 750

Ready to use vaccinia virus expressing Ruc-GFP, mTYR, and GusA

  • Description: Replication competent vaccinia virus encoding Renilla luciferase (Ruc)-green fluorescent protein (GFP) fusion, murine tyrosinase (mTYR), and β-glucuronidase (GusA). These additional transcription units are inserted into, and disrupt the function of, the viral F14.5L, J2R (thymidine kinase) and A56R (hemagglutinin) genes, respectively (see diagram below). 
  • Strain: Lister strain
  • Transgenes: green fluorescent protein (GFP), murine tyrosinase (mTYR), β-glucuronidase (GusA), and Renilla luciferase (Ruc)
  • Biosafety Level: 2
  • Titer: Standard (clarified supernatant, at least 5e6 TCID50/ml).
  • Propagation method: Inoculate producer cells (e.g. Vero) at an MOI of 0.1 for 2-3 hours. 48hrs after infection, harvest culture supernatant and cells. Clarify with low-speed centrifugation and resuspend the pellet with 1mM TrisCl (pH 9). Freeze-thaw or sonicate 3 cycles and clarify the supernatant by low-speed centrifugation. Purify through sucrose cushion.  Resuspend the viral pellet in 1mM TrisCl, pH 9.
  • Proposed Use: Ready to use viral stock for virus propagation and cell killing assays.
  • USDA permit required for shipping? Yes. Click here to fill out Application Form VS 16-3 to obtain a USDA APHIS VS 16-6 or 16-6A permit.

    Transgene Validation

    Fluorescence & β-glucuronidase Assay: Vero cells were mock-infected (A-C) or infected with VV(Li)RucGFP-mTYR-GusA (MOI 0.1) (D-F) and imaged at 48 h.p.i for fluorescence (A-E) or β-glucuronidase (C&F).

     

    Tyrosinase Function: Vero cells were mock-infected or infected with VV(Li)-RucGFP-mTYR-GusA at the indicated MOI. After 48 hours, tyrosinase activity was assayed with addition of 50µl of 2mM L-DOPA. Absorbance was measured using a micro plate reader.

    Luciferase Assay: Vero cells were mock-infected or infected with VV(Li)-RucGFP-mTYR-GusA at an MOI of 0.1 or 1. After 48 hours, 1.25 µg of coelenterazine was added to each of the wells and luminescence (RLU) was immediately measured using a microplate reader.

     

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        Use Fewer Animals

        Our Reduction Campaign

        At Imanis, we are committed to promoting the practice of the 3Rs in animal research. Learn how we are decreasing the use of animals and research as well as saving up to 15% on your orders. Continue reading...