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Construction and application of a novel hepatocyte‐directed vector to simultaneous knockdown and overexpression of multiple genes
Author(s) -
Geng Jianlin,
Wei Haiming,
Sun Rui,
Tian Zhigang
Publication year - 2014
Publication title -
liver international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.873
H-Index - 110
eISSN - 1478-3231
pISSN - 1478-3223
DOI - 10.1111/liv.12336
Subject(s) - gene knockdown , reporter gene , biology , gene , enhancer , internal ribosome entry site , gene silencing , viral vector , microbiology and biotechnology , vector (molecular biology) , rna , hepatocyte , rna interference , gene expression , in vitro , ribosome , genetics , recombinant dna
Background & Aims Liver disease, such as malignancy and hepatitis, often correlates with several genetic disorders. We aimed to construct a hepatocyte‐specific vector that could manipulate multiple genes simultaneously. Methods We selected a highly efficient hepatocyte‐specific α‐foetoprotein ( AFP ) enhancer/albumin promoter (an RNA polymerase II promoter) to express our gene of interest and transcribe micro RNA ‐based sh RNA s (sh RNA mir). Multiple sh RNA mirs were assembled together in tandem to enhance the gene‐silencing effect. By employing the AFP enhancer/albumin promoter and inserting an internal ribosome entry site ( IRES ), a hepatocyte‐specific, multi‐reporter vector that overexpressed both β‐galactosidase (LacZ) and DsRed2 while simultaneously knocking down both EGFP and luciferase expression was successfully constructed and functionally tested in vitro . Results The reporter genes in the multireporter vector were easily replaced by immune‐related genes to construct the Multi‐Vector, which overexpressed human interleukin 10 and silenced both CCL 5 and CX 3 CL 1 ( FKN ) simultaneously in vivo ; visualization of DsRed2 coexpressed to monitor vector function in vivo confirmed that the Multi‐Vector was successfully introduced into the host. Simultaneous manipulation of these multiple genes by the Multi‐Vector synergistically inhibited acute liver injury induced by Poly I:C/D‐GalN injection in mice. The multifunctional cassette was also packaged in and successfully delivered by an adenoviral vector. Conclusions We successfully engineered a vector that can simultaneously regulate multiple genes from a single multigene‐containing vector in a hepatocyte‐specific manner, suggesting the possibility that this method could be extensively and practically utilized in liver gene therapy.