title: Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis.
abstract: Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed "cut-and-paste" mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, 27708, USA.
authors.forename: Syandan
authors.initials: S
authors.lastname: Chakraborty
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, 27708, USA.
authors.forename: HaYeun
authors.initials: H
authors.lastname: Ji
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, 27708, USA.
authors.forename: Jack
authors.initials: J
authors.lastname: Chen
authors.affiliation: 1] Department of Biomedical Engineering, Duke University, Durham, North Carolina, 27708, USA [2] Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, 27708, USA [3] Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, 27708, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, 27708, USA.
authors.forename: Kam W
authors.initials: KW
authors.lastname: Leong
mesh_terms: D002877:Chromosomes, Human / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D005164:Factor IX / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics; D019895:Transposases / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D014764:Viral Proteins; D005164:Factor IX; D013937:Thymidine Kinase; D019895:Transposases