Reversible membrane permeabilization of mammalian cells treated with digitonin and its use for inducing nuclear reprogramming by Xenopus egg extracts.
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.
Miyamoto
Kei
K
Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan.
Yamashita
Teruyoshi
T
Tsukiyama
Tomoyuki
T
Kitamura
Naoya
N
Minami
Naojiro
N
Yamada
Masayasu
M
Imai
Hiroshi
H
D000818:Animals; D002417:Cattle; D002457:Cell Extracts / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002474:Cell-Free System / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D065150:Cellular Reprogramming; D004072:Digitonin / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000378:metabolism; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D010063:Ovum / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D013552:Swine; D014982:Xenopus laevis / Q000378:metabolism
D002457:Cell Extracts; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; D055748:SOXB1 Transcription Factors; D004072:Digitonin