Oligopeptide-mediated gene transfer into mouse corneal endothelial cells: expression, design optimization, uptake mechanism and nuclear localization.
Gene transfer to the corneal endothelium has potential in preventing corneal transplant rejection. In this study, we transfected mouse corneal endothelial cells (MCEC) with a class of novel arginine-rich oligopeptides. The peptides featured a tri-block design and mediated reporter gene expression in MCEC more efficiently than the commercial polyethylenimine standard. The functionality of each block was demonstrated to critically influence the performance of the peptide. Results from confocal imaging and flow cytometry then showed that energy-dependent endocytosis was the dominant form of uptake and multiple pathways were involved. Additionally, uptake was strongly dependent on interactions with cell-surface heparan sulphate. Fluorescence resonance energy transfer studies revealed that the peptide/DNA entered cells as an associated complex and some will have dissociated by 8.5 h. Large-scale accumulation of uncondensed DNA within the nucleus can also be observed by 26 h. Finally, as a proof of biological relevance, we transfected MCEC with plasmids encoding for the functional indoleamine 2,3-dioxygenase (IDO) enzyme. We then demonstrated that the expressed IDO could catalyse the degradation of l-tryptophan, which in turn suppressed the growth of CD4+ T-cells in a proliferation assay.
Seow
Wei Yang
WY
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore.
Yang
Yi-Yan
YY
George
Andrew J T
AJ
D000818:Animals; D001120:Arginine / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004728:Endothelium, Corneal / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry*; D014162:Transfection
D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D009842:Oligopeptides; D004247:DNA; D001120:Arginine
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