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A single plasmid transfection that offers a significant advantage associated with puromycin selection, fluorescence-assisted cell sorting, and doxycycline-inducible protein expression in mammalian cells.
MedLine Citation:
PMID:  21553115     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
Although there are several inducible expression systems for mammalian cells, the most reliable one is the tetracycline-regulated expression system. This system is well-established and widely used by many researchers. Although Clontech provides several types of cells that stably express reverse tetracycline transactivator (rtTA), the cells that are not provided can be generated with pTet-On-Advanced by first integrating this plasmid into the require type of cell and then introducing the genes of interest. These processes are experimental bottlenecks. To improve this situation, we synthesized an all-in-one vector, termed pMAK17, which enables constitutive expression of puromycin N-acetyltransferase, modified Discosoma red fluorescent protein, and rtTA, as well as P(Tight)-driven enhanced green fluorescent protein (EGFP). The pMAK17-transfected cells could be successfully induced to express EGFP, were selectable by fluorescence-activated cell sorting, and displayed puromycin resistance.
Authors:
Takayuki Iwaki; Kazuo Umemura
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Publication Detail:
Type:  Journal Article     Date:  2011-05-07
Journal Detail:
Title:  Cytotechnology     Volume:  63     ISSN:  0920-9069     ISO Abbreviation:  Cytotechnology     Publication Date:  2011 Aug 
Date Detail:
Created Date:  2011-07-22     Completed Date:  2011-11-10     Revised Date:  2012-09-25    
Medline Journal Info:
Nlm Unique ID:  8807027     Medline TA:  Cytotechnology     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  337-43     Citation Subset:  -    
Affiliation:
Department of Pharmacology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka, 431-3192, Japan, tiwaki@hama-med.ac.jp.
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