title: An easy method for preparation of Cre-loxP regulated fluorescent adenoviral expression vectors and its application for direct reprogramming into hepatocytes.
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.
authors.affiliation: Gene Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
authors.forename: Chitose
authors.initials: C
authors.lastname: Kurihara
authors.affiliation: Gene Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Nakade
authors.affiliation: Gene Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan; Institute of Animal Husbandry and Veterinary, Zhejiang Academy of Agricultural Sciences, 198, Shiqiao Rd., Hangzhou, Zhejiang, PR China.
authors.forename: Jianzhi
authors.initials: J
authors.lastname: Pan
authors.affiliation: Institute of Animal Husbandry and Veterinary, Zhejiang Academy of Agricultural Sciences, 198, Shiqiao Rd., Hangzhou, Zhejiang, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Huang
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1, Rue Laurent Fries, Illkirch Cedex 67404, France.
authors.forename: Bohdan
authors.initials: B
authors.lastname: Wasylyk
authors.affiliation: Gene Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Obata
keywords: Adenoviral vector; Cre-loxP; Diffrentiation; Direct reprogramming; Fluorescent protein; Induced- hepatocyte