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本帖最后由 细胞海洋 于 2012-12-6 08:38 编辑 $ _' r; Y/ H" n; o. j
7 h3 R. i, [. [( f" w9 XBOR Papers in Press. Published on January 21, 2009 as DOI:10.1095/biolreprod.108.073676
7 O0 e5 g, O2 h& B8 h. P4 _Copyright 20090 w3 }9 W8 a& }% `0 L# W8 |' ?; F
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Cell-Free Extracts from Mammalian Oocytes Partially Induce Nuclear Reprogramming$ X+ q( @# |* r! r! Z5 |' V5 A8 R2 i' E
in Somatic Cells1
, z% N+ i: l1 N+ b$ sKei Miyamoto3, Tomoyuki Tsukiyama3, Yang Yang4,5, Ning Li4, Naojiro Minami3, Masayasu
/ K2 i3 g. e$ q; K2 E4 z6 rYamada3, and Hiroshi Imai2,3
" K! [1 d" D: P/ O0 h9 uLaboratory of Reproductive Biology3, Graduate School of Agriculture, Kyoto University, Kitashirakawa,
7 M! B3 z( G% I8 a8 Y0 vKyoto 606-8502, Japan) \4 R; s% w. s9 X7 v
State key Laboratory for Agrobiotechnology4, China Agricultural University, Beijing 100139, China
8 J5 V3 T4 q1 [# r$ _( g5 h) dDairy and Animal Science Department5, The Pennsylvania State University, University Park, PA, 16802
& n) P+ E, J8 N5 }! V! A7 k4 Q1Supported by grants from the Japan Society for the Promotion of Science (Research Fellowship. k; n! _2 R" G( b$ e
Program) to K.M., the Ministry of Agriculture, Forestry and Fisheries (Research Project for Utilizing, I0 l1 H3 i# W8 x9 j9 T
Advanced Technologies in Agriculture, Forestry and Fisheries) to H.I., and the National Major Basic
9 B2 w0 S1 U* x3 e1 f0 ]Research Program of China to N.L.
% B1 b2 B) G P2Correspondence: Hiroshi Imai, Ph.D., Laboratory of Reproductive Biology, Graduate School of
; k& e8 d; I+ Z" I8 s& E; t+ D, KAgriculture, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japan. Telephone: 81-75-753-6058; FAX:0 D5 {; m4 L' X
81-75-753-6329; e-mail: imai@kais.kyoto-u.ac.jp
: a6 N0 I/ n; U i7 g: Z# L. v7 a7 |" hShort title: Reprogramming in oocyte cell-free system9 x# E9 L) }7 A$ P6 C! T
Short Sentence: Porcine oocyte extracts were produced from GV and MII oocytes and reprogramming. x0 L9 R. _$ H3 F' z
events such as epigenetic modification, DNA demethylation and activation of pluripotent marker genes% i d% L2 ~( x( @, p; ?9 K
were induced to reversibly permeabilized cells by the extracts.
# ]7 G. U: H" h" } m, {- hKeywords: Reprogramming, oocyte, cell-free system, extracts, pig, histone acetylation, DNA methylation,
5 ]3 E4 s- o# g5 \5 Y0 |" s3 L |NANOG, dedifferentiation
. ^9 z) m" T7 p! k9 a4 e. wABSTRACT1 |, |7 j9 w0 n, R4 q1 v, Y8 W
Nuclear transfer has been regarded as the only reliable tool for studying nuclear reprogramming of9 _2 @3 ?+ U2 h. L) @
mammalian somatic cells by oocytes. However, nuclear transfer is not well suited for biochemical- a5 ?- d3 E8 r( y4 n
analyses of the molecular mechanisms of reprogramming. A cell-free system from oocytes is an attractive5 ]2 H0 L0 f. z" F4 d
alternative way to mimic reprogramming in vitro, since a large number of cells can be treated and" q7 @# l" B2 R1 i b
analyzed. Nevertheless, a cell-free system using oocytes has not been developed in mammals. Here, cell1 e3 Y& T7 b, h; i0 u; P( X
extracts from porcine oocytes were prepared and their ability to induce nuclear reprogramming was D* U0 @8 p! U* l- z
evaluated. Extracts from metaphase II (MII) oocytes erased the machinery for regulating gene expression7 v3 p7 E0 o8 c" F. Z$ b b
in reversibly-permeabilized somatic cells. For example, the extracts caused histone deacetylation and the& T1 h8 @/ r# n& G+ L
disappearance of TATA-binding protein from nuclei. However, MII-extract-treated cells did not show any
# j( L% q8 g/ S3 sobvious changes after cell culture. In contrast, extracts from germinal vesicle (GV) oocytes activated: n+ r s" w' F. R& l
pluripotent marker genes, especially NANOG, and induced partial dedifferentiation after cell culture. The
& R( J/ D3 b9 v: D5 n$ oactivation of pluripotent marker genes by GV extracts was associated with histone acetylation that was9 S; }) m7 {" D0 c, }6 U6 W
induced during extract treatment. These results indicate that GV- and MII-oocyte extracts have different
; K0 m7 [# a8 b$ j2 ^roles on nuclear reprogramming. Furthermore, both oocyte extracts induced site-specific demethylation in( V8 B; o: a0 r7 e- n; q
the upstream region of NANOG. These results indicate that cell-free extracts derived from GV- and MIIoocytes
- x0 I+ i) r. O9 f) s% w& ]could be useful for studying the mechanisms involved in nuclear reprogramming.7 g: e/ R/ H* h; _, v. ~* c5 _6 k
BOR Papers in Press. Published on January 21, 2009 as DOI:10.1095/biolreprod.108.073676" z+ d; q7 r6 e( f
Copyright 2009 by The Society for the Study of Reproduction.
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Cell-Free Extracts from Mammalian Oocytes Partially Induce Nuclear Reprogramming.rar
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发表于 2009-6-13 20:49
