RRC ID 35709
Author Moriyama H, Moriyama M, Sawaragi K, Okura H, Ichinose A, Matsuyama A, Hayakawa T.
Title Tightly regulated and homogeneous transgene expression in human adipose-derived mesenchymal stem cells by lentivirus with tet-off system.
Journal PLoS One
Abstract Genetic modification of human adipose tissue-derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.
Volume 8(6)
Pages e66274
Published 2013-1-1
DOI 10.1371/journal.pone.0066274
PII PONE-D-13-00036
PMID 23776652
PMC PMC3680377
MeSH Adipose Tissue / cytology* Blotting, Western Cell Differentiation / physiology* Cytomegalovirus / genetics DNA Primers / genetics Doxycycline / pharmacology Escherichia coli Flow Cytometry Gene Expression Regulation / drug effects Gene Transfer Techniques* Genetic Vectors / genetics* Green Fluorescent Proteins / genetics Green Fluorescent Proteins / metabolism Humans Lentivirus / genetics Mesenchymal Stem Cells / metabolism* Mesenchymal Stem Cells / physiology Microscopy, Fluorescence Peptide Elongation Factor 1 / genetics Promoter Regions, Genetic / genetics Tetracycline Transgenes / genetics Transgenes / physiology
IF 2.74
Times Cited 8
DNA material CSII-EF-RfA (RDB04387) CSII-CMV-RfA (RDB04386) pCAG-HIVgp (RDB04394) pCMV-VSV-G-RSV-Rev (RDB04393).