Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts |
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Authors: | Alireza Moeen Rezakhanlou Darya Habibi Amy Lai Reza B Jalili Christopher J Ong Aziz Ghahary |
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Institution: | (1) Burn and Wound Healing Research Group, Department of Surgery, University of British Columbia, Vancouver, BC, Canada;(2) The Prostate Centre at Vancouver General Hospital, Department of Surgery, University of British Columbia, Vancouver, BC, Canada;(3) The Endocrinology and Metabolism Research Centre, University of Tehran/Medical Sciences, Tehran, Iran;(4) Burn and Wound Healing Research Lab, Rm 350, Jack Bell Research Centre, 2660 Oak Street, Vancouver, BC, Canada, V6H 3Z6; |
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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. |
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