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
|
WOS Category
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
Resource |
DNA material |
CSII-EF-RfA (RDB04387)
CSII-CMV-RfA (RDB04386)
pCAG-HIVgp (RDB04394)
pCMV-VSV-G-RSV-Rev (RDB04393). |