title: Tightly regulated and homogeneous transgene expression in human adipose-derived mesenchymal stem cells by lentivirus with tet-off system.
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.
authors.affiliation: Pharmaceutical Research and Technology Institute, Kinki University, Higashi-Osaka, Osaka, Japan. moriyama@phar.kindai.ac.jp
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Moriyama
authors.forename: Mariko
authors.initials: M
authors.lastname: Moriyama
authors.forename: Kei
authors.initials: K
authors.lastname: Sawaragi
authors.forename: Hanayuki
authors.initials: H
authors.lastname: Okura
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ichinose
authors.forename: Akifumi
authors.initials: A
authors.lastname: Matsuyama
authors.forename: Takao
authors.initials: T
authors.lastname: Hayakawa
mesh_terms: D000273:Adipose Tissue / Q000166:cytology*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology*; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D004926:Escherichia coli; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000378:metabolism* / Q000502:physiology; D008856:Microscopy, Fluorescence; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D013752:Tetracycline; D019076:Transgenes / Q000235:genetics / Q000502:physiology
chemical_list: D017931:DNA Primers; D020648:Peptide Elongation Factor 1; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D013752:Tetracycline; D004318:Doxycycline