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Controlled nonviral gene delivery and expression using stable neural stem cell line transfected with a hypoxia‐inducible gene expression system
Author(s) -
Liu MengLu,
Oh Jin Soo,
An Sung Su,
Pennant William A.,
Kim Hyo Jin,
Gwak SoJung,
Yoon Do Heum,
Kim Keung Nyun,
Lee Minhyung,
Ha Yoon
Publication year - 2010
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1527
Subject(s) - luciferase , neural stem cell , biology , transfection , enhancer , gene delivery , microbiology and biotechnology , reporter gene , genetic enhancement , gene expression , cell culture , transplantation , stem cell , gene , medicine , genetics , surgery
Abstract Background Nonviral ex vivo local gene therapy systems consisting of regulated gene expression vectors and cellular delivery platforms represent a novel strategy for tissue repair and regeneration. We introduced a hypoxia‐regulated plasmid‐based system into mouse neural stem cells (NSCs) as an efficient gene expression and delivery platform for rapid, robust and persistent hypoxic/ischemic‐regulated gene expression in the spinal cord. Methods A synthetic hypoxia‐responsive erythropoietin (Epo) enhancer, the SV40 minimal promoter and the luciferase (Luc) reporter gene were incorporated in a DsRed‐expressing double‐promoter plasmid for cell lipofection and Zeocin‐selection to establish a hypoxia‐regulated stable NSC line (NSC‐Epo‐SV‐Luc). A nonhypoxia‐regulated stable NSC line (NSC‐SV‐Luc) was also established as a control. Results Under the transcriptional regulation of the Epo enhancer, in vitro luciferase expression in NSC‐Epo‐SV‐Luc, but not in NSC‐SV‐Luc, was sensitively augmented according to the strength and duration of the hypoxic stimulus and was quickly down‐regulated to a low basal level after reoxygenation of the hypoxic cells. Furthermore, deoxygenation of the reoxygenated cells clearly enhanced the luciferase activity again. After transplantation into a rat spinal cord injury (SCI) model, only NSC‐Epo‐SV‐Luc showed ischemic injury‐specific luciferase expression Notably, the engineered NSC lines kept the neural differentiation potential and retained the hypoxia‐regulated luciferase expression after differentiation. Conclusions We propose that NSCs engineered with the Epo‐SV‐therapeutic gene will be valuable for developing a controllable stem cell‐mediated nonviral gene therapy for SCI or other central nervous system diseases accompanied with chronic or episodic hypoxic/ischemic stresses. Copyright © 2010 John Wiley & Sons, Ltd.