title: Insertion of a GFP reporter gene in influenza virus.
abstract: The incorporation of a fluorescent reporter gene into a replication-competent influenza A virus (IAV) has made it possible to trace IAV infection in vivo. This protocol describes the process of inserting a green fluorescent protein (GFP) reporter into the IAV genome using the established reverse genetics system. The strategy begins with the reorganization of segment eight of the IAV genome, during which the open reading frames of nonstructural protein 1 (NS1) and the nuclear export protein (NEP) are separated to allow for GFP fusion to the NS1 protein. The NS1, GFP, and NEP open reading frames (ORF) are then cloned into the IAV rescue system backbone. Upon construction of the GFP-encoding segment eight rescue plasmid, recombinant NS1-GFP influenza virus can be rescued via co-transfection with the remaining seven rescue plasmids. The generated NS1-GFP IAV can subsequently be used to visualize infected cells, both in vitro and in vivo.
authors.affiliation: Department of Microbiology, University of Chicago, Chicago, Illinois.
authors.forename: Jasmine T
authors.initials: JT
authors.lastname: Perez
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York, New York.
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
authors.affiliation: Department of Microbiology, University of Chicago, Chicago, Illinois.
authors.forename: Balaji
authors.initials: B
authors.lastname: Manicassamy
mesh_terms: D019677:Artificial Gene Fusion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D009980:Influenza A virus / Q000235:genetics* / Q000254:growth & development; D059386:Reverse Genetics / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014773:Virology / Q000379:methods
chemical_list: C085820:INS1 protein, influenza virus; D017361:Viral Nonstructural Proteins; D049452:Green Fluorescent Proteins