title: Generation and analysis of lentivirus expressing a 2A peptide-linked bicistronic fluorescent construct.
abstract: This weeklong protocol for making and testing lentivirus has been used in the Advanced Topics in Molecular Neuroscience (ATMN) lecture and laboratory course at Cold Spring Harbor Laboratory (CSHL) for nearly a decade. Lentiviruses are derived from HIV-1 and are ideal vehicles for the delivery of multiple genes of interest into target cells. In this protocol, 2A peptide-linked sequences are used to create a bicistronic lentiviral construct containing a ubiquitous promoter (chick β actin with a cytomegalovirus [CMV] early enhancer) driving dual expression of two fluorescent proteins (FP): H2B-Cerulean (a nuclear-localized blue FP) and Dendra2 (a photoactivatable green FP that converts to red after exposure to UV light). Polymerase chain reaction (PCR) amplification of the bicistronic insert is followed by subcloning into a lentiviral vector and transfection into a packaging cell line. The resulting viral supernatants can be used to prepare concentrated stocks and infect cells for imaging via epifluorescent and confocal microscopy.
authors.affiliation: Keck School of Medicine, University of Southern California, Los Angeles, California 90033; Children's Hospital Los Angeles, Los Angeles, California 90027.
authors.forename: David
authors.initials: D
authors.lastname: Huss
authors.affiliation: Keck School of Medicine, University of Southern California, Los Angeles, California 90033; Children's Hospital Los Angeles, Los Angeles, California 90027.
authors.forename: Rusty
authors.initials: R
authors.lastname: Lansford
mesh_terms: D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004247:DNA / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D009841:Oligonucleotides / Q000235:genetics; D010455:Peptides / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014162:Transfection; D014170:Transformation, Genetic
chemical_list: D009841:Oligonucleotides; D010455:Peptides; D049452:Green Fluorescent Proteins; D004247:DNA