Green fluorescent protein expressed by a recombinant vaccinia virus permits early detection of infected cells by flow cytometry.
We have tested Green Fluorescent Protein (GFP) expressed by a vaccinia virus recombinant as a marker for viral infection. Virus recombinants expressing either wild-type GFP, or a Ser65 to Thr mutated version (GFP-S65T) were used to infect cultured cells, and the appearance of fluorescence was followed during infection by flow cytometry. Although both versions were detectable in infected cells, GFP-S65T gave up to 26-fold brighter fluorescence than wild-type GFP when excited by an argon laser beam (488 nm). In addition, GFP-S65T fluorescence appeared earlier, and infected cells could be detected above background as soon as 1 h after infection. We have used this construct to infect porcine peripheral blood lymphocytes, and show its usefulness to study virus tropism when used in combination with cell-type specific markers. Thus, GFP provides a direct, fast and convenient way to monitor infection by flow cytometry.
Domínguez
J
J
Departamento de Sanidad Animal, Centro de Investigación en Sanidad Animal-INIA, Madrid, Spain.
Lorenzo
M M
MM
Blasco
R
R
D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D005470:Fluorometry; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D008214:Lymphocytes / Q000737:chemistry / Q000821:virology*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D013552:Swine; D014616:Vaccinia virus / Q000235:genetics / Q000302:isolation & purification*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins