Cloning and transient expression of cytoprotective factor, HO-1, in mesenchymal stem cells using the adenoviral expression system through Gateway technology

Heme oxegenase1 (HO-1) is one of the potent cytoprotective factors. The goal of this study was to perform cloning and transient over expression of the human HO-1 gene in mesenchymal stem cells (MSCs) using the adenoviral expression system based on the gateway technology. In order to induce expression of HO-1، A549 cell lines were exposed to UV for 1 hour. The full length cDNA of HO-1 was isolated and cloned into pENTR TOPO/D vector by TOPO cloning reaction. To construct the expression clone، a LR recombination reaction was carried out between the entry clone and destination vector، pAd/CMV/V5-DEST. The recombinant virus was produced in the appropriate mammalian cell line. MSCs were infected by the recombinant virus expressing HO-1. The results showed that human recombinant HO-1 was successfully cloned and the accuracy of the gene and its frame in the vector were confirmed by DNA sequencing. Expression of HO-1 in MSCs was confirmed by RT-PCR and western blot analysis. The results indicated that the expression of HO-1 is transient. Transient expression of human HO-1 gene in MSCs by using adenovirus expression system may be considered as an efficient gene transfer strategy into MSCs in order to promote stem cell therapy.