Puromycin resistance (pac) gene as a selectable marker in vaccinia virus.
The antibiotic puromycin, an inhibitor of protein synthesis, was shown to inhibit vaccinia virus (VV) replication. We evaluated the use of puromycin-resistance (pac) gene as a selectable marker in VV. A recombinant vaccinia virus expressing pac (VV-pac) under the control of a viral early/late promoter was constructed and characterized. VV-pac grew in the presence of puromycin at concentrations that were inhibitory for the parental VV and toxic for the cells. Isolation of recombinant VV usually relies on plaque purification under selective conditions. Because virus plaquing was not feasible under inhibitory puromycin concentration, a protocol based on serial passage of virus was devised. The usefulness of this procedure in selecting pac expressing viruses was tested by isolating a recombinant VV.
Sánchez-Puig
J M
JM
Departamento de Mejora genética y biotecnología-I.N.I.A., km 7, E-28040, Ctra. La Coruña, Spain.
Blasco
R
R
D000818:Animals; D002460:Cell Line; D004274:DNA, Recombinant; D004305:Dose-Response Relationship, Drug; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011691:Puromycin / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013997:Time Factors; D014616:Vaccinia virus / Q000187:drug effects / Q000235:genetics*; D014779:Virus Replication / Q000187:drug effects
D004274:DNA, Recombinant; D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011691:Puromycin