• 0.25mL / Standard / Y ((OV4002-STAN)) $ 1,500

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.