Document Detail

Reversible membrane permeabilization of mammalian cells treated with digitonin and its use for inducing nuclear reprogramming by Xenopus egg extracts.
MedLine Citation:
PMID:  19049416     Owner:  NLM     Status:  MEDLINE    
Plasma membranes can be reversibly permeabilized by Streptolysin O. The permeabilized cells can be reprogrammed and partially dedifferentiated in the cell-free system from egg extracts. However, the permeabilizing activity of Streptolysin O is not stable, and therefore it is difficult to control its activity. An alternative method for reversible permeabilization is useful for establishing a cell-free system. Here, we used a nonionic detergent, digitonin, for permeabilization. A low concentration of digitonin induced reversible permeabilization of the plasma membrane in bovine, mouse, and porcine somatic cells. The permeabilized cells were treated with Xenopus laevis egg extracts. The treated cells showed exchange of nuclear proteins from extracts such as incorporation of Xenopus-specific histone B4 and Lamin LIII into nuclei. After resealing of the membrane, the cells showed upregulation of OCT4, SOX2, and NANOG expression. Our results suggest that reversible permeabilization with digitonin can be used to induce nuclear reprogramming and to activate pluripotent genes by a cell-free system.
Kei Miyamoto; Teruyoshi Yamashita; Tomoyuki Tsukiyama; Naoya Kitamura; Naojiro Minami; Masayasu Yamada; Hiroshi Imai
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Cloning and stem cells     Volume:  10     ISSN:  1557-7457     ISO Abbreviation:  Cloning Stem Cells     Publication Date:  2008 Dec 
Date Detail:
Created Date:  2008-12-03     Completed Date:  2009-02-02     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101125444     Medline TA:  Cloning Stem Cells     Country:  United States    
Other Details:
Languages:  eng     Pagination:  535-42     Citation Subset:  IM    
Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan.
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MeSH Terms
Cell Extracts / pharmacology
Cell Membrane / drug effects,  metabolism
Cell Membrane Permeability / drug effects*
Cell Nucleus / drug effects,  metabolism
Cell-Free System / drug effects,  physiology*,  ultrastructure
Digitonin / pharmacology*
Fibroblasts / cytology,  metabolism
Gene Expression
Homeodomain Proteins / metabolism
Nuclear Reprogramming*
Octamer Transcription Factor-3 / metabolism
Ovum / drug effects,  physiology*,  ultrastructure
Pluripotent Stem Cells / cytology,  metabolism*
SOXB1 Transcription Factors / metabolism
Xenopus laevis / metabolism
Reg. No./Substance:
0/Cell Extracts; 0/Homeodomain Proteins; 0/Octamer Transcription Factor-3; 0/SOXB1 Transcription Factors; 11024-24-1/Digitonin

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