title: Comparative analysis of molecular strategies attenuating positional effects in lentiviral vectors carrying multiple genes.
abstract: Efficient, high-level expression of multiple genes is often difficult to achieve in retroviral vectors, due to positional effects affecting transcription of adjacent sequences. Here we describe the comparative analysis of different strategies for co-expressing two model cDNA sequences in the context of a second generation lentiviral vector system. A first option was based on the generation of a polycistronic construct by subcloning an internal ribosome entry site (IRES) sequence between tandem cDNAs. IRES-dependent translation of the cDNA placed downstream (3') of the first transgene was poor, and the protein was barely detectable in transduced cells. A similar result was obtained when both transgenes were placed under the transcriptional control of two independent internal promoters. When these independent transcription units were separated by the 5'HS4 chromatin insulator of the chicken beta-globin locus, a marked increase of the expression of the downstream protein was observed. Similarly, insertion of a polyadenylation sequence between the tandem transcription units fully restored - in transfection experiments - the expression of the downstream sequence, whose protein pattern was identical to the single-gene control, suggesting that in this specific construct transcriptional interference was the likely cause of the observed positional effects. These results indicate that chromatin insulator sequences can be useful molecular tools to overcome positional effects in the context of lentiviral vectors.
authors.affiliation: Department of Structural and Functional Biology, University of Insubria, Busto Arsizio 21052, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Osti
authors.forename: Emanuela
authors.initials: E
authors.lastname: Marras
authors.forename: Isabella
authors.initials: I
authors.lastname: Ceriani
authors.forename: Greta
authors.initials: G
authors.lastname: Grassini
authors.forename: Tiziana
authors.initials: T
authors.lastname: Rubino
authors.forename: Daniela
authors.initials: D
authors.lastname: Viganò
authors.forename: Daniela
authors.initials: D
authors.lastname: Parolaro
authors.forename: Gianpaolo
authors.initials: G
authors.lastname: Perletti
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D038101:Insulator Elements / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D039104:RNA 3' Polyadenylation Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D014158:Transcription, Genetic
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins