Abstract
The recombinant adenoviral gene expression system is a powerful tool for gene delivery. However, it is difficult to obtain high titers of infectious virus, principally due to the toxicity of the expressed gene which affects on virus replication in the host HEK293 cells. To avoid these problems, we generated a Cre-loxP-regulated fluorescent universal vector (termed pAxCALRL). This vector produces recombinant adenoviruses that express the red fluorescent protein (RFP) instead of the inserted gene during proliferation, which limits toxicity and can be used to monitor viral replication. Expression of the gene of interest is induced by co-infection with an adenovirus that expresses Cre-recombinase (AxCANCre). Recombinant adenovirus produced by this system that express Hnf4α and Foxa2 were used to reprogram mouse embryo fibroblast (MEF) into induced-hepatocyte-like cells (iHep) following several rounds of infection, demonstrating the efficacy of this new system.
Highlights
Various virus-based vector systems are used for gene transfer, including adenoviruses, lentiviruses, retroviruses and adenoassociated viruses
Adenovirus type 5 (Ad5) is generally considered to be non-pathogenic and is often used for gene transduction, a recent report suggests that it induces obesity [22]
As the budded adenovirus particles tends to infect neighbouring cells, the virus producing cells can be observed as red fluorescent protein (RFP) positive colonies
Summary
Various virus-based vector systems are used for gene transfer, including adenoviruses, lentiviruses, retroviruses and adenoassociated viruses. Adenovirus type 5 (Ad5) is generally considered to be non-pathogenic and is often used for gene transduction, a recent report suggests that it induces obesity [22] Adenovirus can infect both actively dividing and terminally differentiated cells [4] and does not require integration into the host genome for gene expression [23]. It was reported that chemical coupling of adenovirus with a peptide that has a high affinity for a hepatic stellate cell (HSC) surface protein drastically facilitates infection of the corresponding cells [16] Using this method, Reetz et al directly reprogrammed hepatic myofibroblasts into hepatocytes in vivo by introducing an adenoviral vector that expresses Foxa, Gata, Hnf1a, and Hnf4a [20]. Our Cre-loxP regulated fluorescent adenoviral expression vector can be expected to produce higher viral titers, as it expresses RFP during viral proliferation instead of the inserted potentially-toxic gene. Thse adenoviral vectors can be used for successful direct reprogramming of mouse embryo fibroblasts (MEF) into hepatocyte-like cells [called induced-Hepatocyte (iHep) by Sekiya et al.] [19]
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