title: Generation of recombinant vaccinia viruses via green fluorescent protein selection.
abstract: We developed a rapid method to generate recombinant vaccinia viruses (rVVs) based upon a bicistronic cassette encoding the gene for green fluorescent protein (GFP) and a foreign gene of interest separated by an internal ribosome entry site (IRES). As proof-of-concept, we inserted a mutant env gene of human immunodeficiency virus (HIV) into the cassette, which was cloned into the vaccinia virus (VV) insertion vector pSC59 under the control of the early-late VV synthetic promoter and flanked by disrupted tk gene sequences. To generate rVVs, 293T cells were inoculated with wild-type (wt) VV, followed by transfection of the modified pSC59 vector containing the bicistronic cassette, which allows expression of GFP and the protein of interest. Next, GFP-positive cells were isolated by flow cytometry or by picking under a fluorescent microscope. Thymidine kinase-deficient (Tk(-)) 143B cells were then exposed to lysates of GFP-positive 293T cells and cultured in the presence of bromodeoxyuridine. This selection allows only Tk(-) rVV to remain viable. We demonstrated the success of this GFP selection strategy by expressing high levels of mutant HIV Env. Our approach shortens the time needed to generate rVVs and represents a practical approach to generate recombinant proteins.
authors.affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
authors.forename: Sergei
authors.initials: S
authors.lastname: Popov
authors.forename: Saied
authors.initials: S
authors.lastname: Mirshahidi
authors.forename: Sosthène
authors.initials: S
authors.lastname: Essono
authors.forename: Ruijiang
authors.initials: R
authors.lastname: Song
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Wang
authors.forename: Ruth M
authors.initials: RM
authors.lastname: Ruprecht
mesh_terms: D002460:Cell Line; D005434:Flow Cytometry; D015752:Genes, env; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006678:HIV / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase