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.

A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells

PICCO, GABRIELE;PETTI, CONSALVO;TRUSOLINO, Livio;BERTOTTI, Andrea;MEDICO, Enzo
Last
2015

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.
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http://www.nature.com/articles/srep14721
Picco, Gabriele; Petti, Consalvo; Trusolino, Livio; Bertotti, Andrea; Medico, Enzo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1529850
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