title: A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo.
abstract: The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.
authors.affiliation: Department of Biology, McGill University, Montréal, Quebec, Canada.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Geiling
authors.forename: Guillaume
authors.initials: G
authors.lastname: Vandal
authors.forename: Ada R
authors.initials: AR
authors.lastname: Posner
authors.forename: Angeline
authors.initials: A
authors.lastname: de Bruyns
authors.forename: Kendall L
authors.initials: KL
authors.lastname: Dutchak
authors.forename: Samantha
authors.initials: S
authors.lastname: Garnett
authors.forename: David
authors.initials: D
authors.lastname: Dankort
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D015415:Biomarkers; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53