An easy method for preparation of Cre-loxP regulated fluorescent adenoviral expression vectors and its application for direct reprogramming into hepatocytes

- We generated Cre-loxP-regulated fluorescent universal adenoviral vector system.
- The replication of recombinant virus produced by this system is easy to be monitored.
- This system gives higher titer and faster replication of virus in some cases.
- The expression of inserted gene is induced by co-infection of Cre-expression vector.
- We confirmed this system works for the direct reprogramming of MEF into Hepatocyte.

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.