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This is a method to generate “scarless” recombinant vaccinia viruses using host-range selection and visual identification of recombinant viruses.
Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV’s easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Several methods have been developed to generate recombinant VACV, including marker selection methods and transient dominant selection. Here, we present a refinement of a host range selection method coupled with visual identification of recombinant viruses. Our method takes advantage of selective pressure generated by the host antiviral protein kinase R (PKR) coupled with a fluorescent fusion gene expressing mCherry-tagged E3L, one of two VACV PKR antagonists. The cassette, including the gene of interest and the mCherry-E3L fusion is flanked by sequences derived from the VACV genome. Between the gene of interest and mCherry-E3L is a smaller region that is identical to the first ~150 nucleotides of the 3’ arm, to promote homologous recombination and loss of the mCherry-E3L gene after selection. We demonstrate that this method permits efficient, seamless generation of rVACV in a variety of cell types without requiring drug selection or extensive screening for mutant viruses.
Vaccinia virus (VACV) was instrumental for the first successful eradication of a human pathogen, variola virus (VARV), from nature. Ever since the extermination of variola virus, poxviruses including VACV have continued to be useful therapeutic viruses for both human and animal medicine. For example, a VACV-based rabies virus vaccine has been very effective in preventing transmission of sylvatic rabies in Europe1 and the United States2. More recently, recombinant poxviruses expressing a variety of anti-tumor molecules (e.g., single-chain antibodies or human erythropoietin) have seen encouraging success as oncolytic agent....
1. Generating the recombination vector
We used the procedure diagrammed in Figure 1 to generate a VACV lacking both PKR antagonists E3L and K3L, by replacing E3L with EGFP in a virus already deleted for K3L (vP872). Figure 2 shows red fluorescent plaques in PKR competent RK13 cells indicative of viral expression of mCherry-E3L, as well as EGFP expressed in RK13+E3L+K3L cells confirming the loss of E3L and collapse of the mCherry-E3L selection marker. Figure 3 confirms th.......
Here we present a variation of a transient marker selection strategy 32 to generate recombinant vaccinia viruses without retaining foreign DNA in the final recombinant virus. Our strategy uses selective pressure mediated by the host antiviral protein PKR rather than other forms of selective pressure such as antibiotics. The use of host antiviral genes eliminates the possibility of chemically induced phenotypic changes in the cells, or increased risk of mutation due to selection drugs. Furthermore,.......
This project was funded by the National Institutes of Health (AI114851) to SR.
....Name | Company | Catalog Number | Comments |
2X-Q5 Master Mix | NEB | M0492L | High-fidelity polymerase used in PCR |
Ampicillin | ThermoFisher Scientific | 11593027 | Bacterial selective agent |
Disposable Cell Scrapers | ThermoFisher Scientific | 08-100-242 | Cell scraper to harvest infected cells |
EVOS FL Auto 2 Cell imaging system | ThermoFisher Scientific | AMAFD2000 | Fluorescent microscope |
EVOS Light Cube, GFP | ThermoFisher | AMEP4651 | GFP Cube |
EVOS Light Cube, RFP | ThermoFisher | AMEP4652 | RFP Cube |
GenJet | SignaGen Laboratories | SL100489 | Transfection reagent |
Luria Bertani (LB) Broth | Gibco | 10855021 | Bacterial growth medium |
Monarch DNA gel extraction kit | NEB | T1020L | Gel purification kit used to purify amplicons and linearized vectors |
Monarch Plasmid Miniprep kit | NEB | T1010L | Miniprep kit ussed to purify plasmids |
NanoDrop One | ThermoFisher Scientific | ND-ONE-W | Spectrophotometer used to measure RNA and DNA concentration |
NEBuilder Master Mix | NEB | E2621L | Isothermal enzymatic assembly kit used to generate the recombination vector |
Q500 Sonicator | Qsonica | Q500-110 | Sonicator for virus lysates |
RK13 cells | ATCC | CCL-37 | Rabbit kidney cells |
VWR Multiwell Cell Culture plates | VWR | 10062-892 | Cell culture plates |
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