title: Highly efficient stable expression of indoleamine 2,3 dioxygenase gene in primary fibroblasts.
abstract: Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.
authors.affiliation: Burn and Wound Healing Research Group, Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Alireza Moeen
authors.initials: AM
authors.lastname: Rezakhanlou
authors.affiliation: Burn and Wound Healing Research Group, Department of Surgery, University of British Columbia, Vancouver, BC, Canada.|The Prostate Centre at Vancouver General Hospital, Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Darya
authors.initials: D
authors.lastname: Habibi
authors.affiliation: Burn and Wound Healing Research Group, Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Amy
authors.initials: A
authors.lastname: Lai
authors.affiliation: Burn and Wound Healing Research Group, Department of Surgery, University of British Columbia, Vancouver, BC, Canada.|The Endocrinology and Metabolism Research Centre, University of Tehran/Medical Sciences, Tehran, Iran.
authors.forename: Reza B
authors.initials: RB
authors.lastname: Jalili
authors.affiliation: The Prostate Centre at Vancouver General Hospital, Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ong
authors.affiliation: Burn and Wound Healing Research Group, Department of Surgery, University of British Columbia, Vancouver, BC, Canada.|Burn and Wound Healing Research Lab, Rm 350, Jack Bell Research Centre, 2660 Oak Street, Vancouver, BC, Canada, V6H 3Z6.
authors.forename: Aziz
authors.initials: A
authors.lastname: Ghahary