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本帖最后由 细胞海洋 于 2012-12-6 08:38 编辑
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BOR Papers in Press. Published on January 21, 2009 as DOI:10.1095/biolreprod.108.073676
1 n# S( g+ X; O/ T BCopyright 2009
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Cell-Free Extracts from Mammalian Oocytes Partially Induce Nuclear Reprogramming
2 q7 }, }0 p3 X8 C8 I2 `( A* o, x5 [in Somatic Cells1
8 j+ A' o9 x8 a( _% v+ ^Kei Miyamoto3, Tomoyuki Tsukiyama3, Yang Yang4,5, Ning Li4, Naojiro Minami3, Masayasu
+ @5 j/ J/ M3 `( ]8 uYamada3, and Hiroshi Imai2,32 v' p* ?' ], g9 b, F
Laboratory of Reproductive Biology3, Graduate School of Agriculture, Kyoto University, Kitashirakawa,' n5 t8 A0 v3 H) R7 }. r- y2 r
Kyoto 606-8502, Japan' |9 Z' }0 Y9 I" m$ [8 F1 I
State key Laboratory for Agrobiotechnology4, China Agricultural University, Beijing 100139, China9 d1 J' S( g9 r' h
Dairy and Animal Science Department5, The Pennsylvania State University, University Park, PA, 16802
/ J! Y% z2 f+ x, z# k! q1Supported by grants from the Japan Society for the Promotion of Science (Research Fellowship& F& @3 O0 G' `) m8 R v" J
Program) to K.M., the Ministry of Agriculture, Forestry and Fisheries (Research Project for Utilizing+ I) Y# e3 P. {1 V
Advanced Technologies in Agriculture, Forestry and Fisheries) to H.I., and the National Major Basic
( E' A. Q/ B9 q( R5 EResearch Program of China to N.L.7 o4 I+ R9 T' C* C
2Correspondence: Hiroshi Imai, Ph.D., Laboratory of Reproductive Biology, Graduate School of
& D% R' s4 t8 Q, ZAgriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan. Telephone: 81-75-753-6058; FAX:
% ^6 s; N) W/ l8 f: s81-75-753-6329; e-mail: imai@kais.kyoto-u.ac.jp$ j1 q6 f `2 s" U& m) a! M% f
Short title: Reprogramming in oocyte cell-free system
6 X* S6 A/ |( Q/ T& wShort Sentence: Porcine oocyte extracts were produced from GV and MII oocytes and reprogramming% ]+ P* M& B" d
events such as epigenetic modification, DNA demethylation and activation of pluripotent marker genes
: o8 A" _3 n; e( y5 L7 ^1 o: swere induced to reversibly permeabilized cells by the extracts./ D0 A( q# p$ C
Keywords: Reprogramming, oocyte, cell-free system, extracts, pig, histone acetylation, DNA methylation,
- Z* i0 y: E+ Z9 ?. ]; V$ D! v' X" ?NANOG, dedifferentiation
$ Q% U6 F4 y) x& |, {6 i5 t/ HABSTRACT1 @9 ?: [7 A7 }4 L" }
Nuclear transfer has been regarded as the only reliable tool for studying nuclear reprogramming of, t* [: }* L& z, I+ [$ |" E
mammalian somatic cells by oocytes. However, nuclear transfer is not well suited for biochemical
! ?% Z. i; }# ^0 W. \ Yanalyses of the molecular mechanisms of reprogramming. A cell-free system from oocytes is an attractive* w* i! } c2 _, j" B( Q s/ J
alternative way to mimic reprogramming in vitro, since a large number of cells can be treated and
% `. q3 I, T% J. F! danalyzed. Nevertheless, a cell-free system using oocytes has not been developed in mammals. Here, cell
9 {' x$ N0 c, q. qextracts from porcine oocytes were prepared and their ability to induce nuclear reprogramming was/ `: L8 R5 \2 V/ ]1 ~
evaluated. Extracts from metaphase II (MII) oocytes erased the machinery for regulating gene expression( ^! x! Q- ~3 F% K! p
in reversibly-permeabilized somatic cells. For example, the extracts caused histone deacetylation and the
: b* X- ~5 O& v( B% _% r6 q G7 pdisappearance of TATA-binding protein from nuclei. However, MII-extract-treated cells did not show any
, W6 W, i, Q2 u& Q) Aobvious changes after cell culture. In contrast, extracts from germinal vesicle (GV) oocytes activated
; Q$ y8 A# L, K' D4 o- x6 H4 C' ?pluripotent marker genes, especially NANOG, and induced partial dedifferentiation after cell culture. The
9 z" f5 J/ M* |- `2 t# l/ Mactivation of pluripotent marker genes by GV extracts was associated with histone acetylation that was: Y% Y' y( `5 f+ E% A
induced during extract treatment. These results indicate that GV- and MII-oocyte extracts have different6 j+ z: Q& t1 L+ x8 X8 ]
roles on nuclear reprogramming. Furthermore, both oocyte extracts induced site-specific demethylation in
% S" O' W$ [+ R3 [( {1 g( ^the upstream region of NANOG. These results indicate that cell-free extracts derived from GV- and MIIoocytes% u K) U# @! W9 ~; w
could be useful for studying the mechanisms involved in nuclear reprogramming.
# s2 b3 D) q" @0 ^9 \0 G; d* NBOR Papers in Press. Published on January 21, 2009 as DOI:10.1095/biolreprod.108.073676) e1 c$ Y5 T/ Z, I6 T U( M- S
Copyright 2009 by The Society for the Study of Reproduction.( }0 p0 R3 E# ^* K& J
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发表于 2009-3-17 09:30
发表于 2009-6-13 20:49
