Abstract
Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.
Highlights
The expression of multiple proteins in eukaryotic cells has become crucial in many biomedical applications of contemporary cell biology [1]
We previously reported that the Foot and Mouth Disease Virus 2A sequence (F2A) technology can be used to link in frame three coding sequences obtaining a single open reading frame of 4.3 Kbp that can be expressed in eukaryotic cells as three discrete protein products [32]
Single gene-expressing vectors have been produced as controls and cells transfected to investigate the contribution of each gene in the downregulation of the inflammatory response. human heme oxygenase 1 (hHO-1), human ecto-5’-nucleotidase (hE5NT) and human ecto-nucleoside triphosphate diphosphohydrolases (hENTPD1) coding sequences were cloned into the same vector backbone used to produce pCX-TRI-2A plasmid (Fig 1)
Summary
The expression of multiple proteins in eukaryotic cells has become crucial in many biomedical applications of contemporary cell biology [1]. The 2A and ‘2A-like’ sequences have been successfully used to express several proteins in lentivirus-mediated gene therapy approaches [5] and in the production of monoclonal antibodies in transgenic mice [6]. Another biomedical application requiring multiple genetic modifications in eukaryotic cells is xenotransplantation, where the complexity of immunological barriers to be overcome for a successful experiment requires several human genes to be overexpressed in the cells of the potential donor species [7,8]. Transplanted organs are subjected to several antigen-independent injuries, such as ischemia/reperfusion injury (IRI) [10,11] and free radicals production [12]
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