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Immunology JoVE (Open Access) Citable · DOI

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

DOI: 10.3791/51151-v
What you'll learn
  • ✓ Generate recombinant vaccinia viruses with fluorescent protein tags using transient dominant selection
  • ✓ Design and construct modular recombinant vectors for VACV protein expression
  • ✓ Validate viral purity and recombinant status through plaque assay and PCR screening
  • ✓ Apply rapid, efficient protocols for simultaneous multi-protein fluorescent tagging
Protocol

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently tagged proteins simultaneously using the method of transient dominant selection is described here.

Difficulty
advanced
Total time
~5–7 days (vector construction through viral purification and screening)
Biosafety
BSL-2

Steps

1
Design and construct recombinant expression vector

Design modular vaccinia virus vector incorporating fluorescent protein tags and selection markers. Clone target genes into the vector backbone to enable simultaneous expression of tagged proteins.

▶ 01:34
2
Generate recombinant vaccinia virus by transfection

Transfect mammalian cells with recombinant vector plasmid. Use transient dominant selection methodology to enrich for cells containing integrated recombinant viral genomes during successive viral passages.

▶ 02:48
3
Isolate and expand viral clones by plaque assay

Perform plaque assay on infected cells to identify and isolate clonal viral populations without selection pressure, enabling recovery of pure recombinant virus stocks.

▶ 05:26
4
Verify recombinant status using PCR screening

Extract genomic DNA from viral stocks and perform PCR using gene-specific primers to confirm successful incorporation of fluorescent protein sequences and validate viral purity.

▶ 05:59
5
Analyze recombinant vectors and viral genome integration

Examine PCR results and characterize successful recombinant vector construction and integration into VACV genome across multiple viral clones.

▶ 07:23
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