title: A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells.
abstract: We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.
authors.affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.|University of Torino, Department of Oncology, SP 142, Km 3.95, 10060 Candiolo, Torino, Italy.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Picco
authors.affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.|University of Torino, Department of Oncology, SP 142, Km 3.95, 10060 Candiolo, Torino, Italy.
authors.forename: Consalvo
authors.initials: C
authors.lastname: Petti
authors.affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.|University of Torino, Department of Oncology, SP 142, Km 3.95, 10060 Candiolo, Torino, Italy.
authors.forename: Livio
authors.initials: L
authors.lastname: Trusolino
authors.affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.|University of Torino, Department of Oncology, SP 142, Km 3.95, 10060 Candiolo, Torino, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bertotti
authors.affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.|University of Torino, Department of Oncology, SP 142, Km 3.95, 10060 Candiolo, Torino, Italy.
authors.forename: Enzo
authors.initials: E
authors.lastname: Medico
mesh_terms: D000818:Animals; D002460:Cell Line; D004167:Diphtheria Toxin / Q000633:toxicity*; D004351:Drug Resistance / Q000235:genetics*; D015870:Gene Expression; D020868:Gene Silencing; D005822:Genetic Vectors; D064593:Heterografts; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D011506:Proteins / Q000235:genetics; D014161:Transduction, Genetic; D014162:Transfection; D019076:Transgenes
chemical_list: C115726:DPH2 protein, human; D004167:Diphtheria Toxin; D035683:MicroRNAs; D011506:Proteins