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我把内容放出来，请各位分析一下，谢谢
( m" W( G! j$ z7 h' m* W( z2 VCell culture
. | `: I6 D# p! kOG2-MEFs were cultured on gelatin-coated dishes in normal MEF media: high-glucose D-MEM
! a; m: Y; E3 n U" ~- l(Invitrogen) with 10% FBS, 0.1 mM non-essential amino acids, and 2 mM L-glutamine. piPS5 Q |* n- c4 N$ n: C$ J
cells were cultured on irradiated CF1 MEFs with normal mESC growth media, which consist of
5 m3 u* `2 I7 G+ J8 Z) P* m- X! u* WKnockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 mM 2-ME, 2 mM L-glutamine,
) {9 Y" C* b0 p1 D" S4 E0.1 mM non-essential amino acids, and 1000 units/ml LIF (ESGRO, Chemicon International). The' O, p2 t4 z5 o, U
piPS cells were passaged every 3 days as a single cell suspension using 0.05% trypsin/EDTA
9 E2 Q3 A0 [9 U, Yand seeded at 1.0x104 cells per cm2 for routine culture. For feeder-free culture, cells are grown
- I- Z) Z' p5 `: g, lon gelatin-coated tissue culture dishes in chemically defined media, which consist of Knockout
6 Z% i3 `' N% a1 ^2 DDMEM supplemented with 1xN2, 1xB27, 0.1 mM 2-ME, 2 mM L-glutamine, 0.1 mM nonessential6 c( c, g% T8 Q$ O- E7 N; d3 g
amino acids, 50 μg/ml BSA fraction V (GIBCO), 103 units/ml LIF and 10 ng/ml BMP4
* w5 [+ B7 n- I( m! g3 U(R&D).
8 ^3 d' I8 D0 i9 @4 r" T, T; gGeneration of piPS cells" r% p4 B# {% A( J
OG2-MEFs were seeded at 5x104 cells per well in a 6-well plate coated with gelatin in normal' |1 U. G y6 N+ a. Z9 P l' _# o$ ?
MEF media (DMEM supplemented with 10% FBS). On the next day, media was changed to the
' d/ }1 t/ B8 C e0 O0 ^protein transduction media, which were prepared by mixing the recombinant reprogramming
7 s) J: j$ {& J& }* X6 R& zproteins at the final concentration of 8 μg/ml with regular mES cell growth medium
4 \4 j# f1 \# ksupplemented with 1000 units/ml LIF. After overnight culture in the protein transduction media,) U6 b3 j& Z8 X% z( O& l
media was changed to normal mESC growth media, and cells were cultured for additional 36
1 p8 [1 @7 F! R) bhours before repeating the same protein transduction cycle. Total four protein transduction+ r+ [7 {2 ~* }1 \7 x1 W; p
cycles were applied on the cells. After completing protein transduction, cells were then7 o6 V! U! X. c
passaged onto irradiated CF-1 MEF feeder cells at day 9 in normal mESC growth media. Media+ N8 J% r: T2 k+ ]5 x2 p( @
were changed every 3-4 days until GFP+ colonies were observed around day 30-35. GFP+
' I3 A [8 R9 X0 Jcolonies were then passaged onto new irradiated MEF feeder cells in normal mESC growth
7 q# X1 `$ u( K8 s/ imedia, and stably maintained and expanded as piPS cells. Some colonies were further selected
% R6 B9 `5 {8 E9 X8 Jand expanded in the presence of pluripotin (1 μM) or PD0325901 (1 μM). |
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发表于 2012-3-25 09:33
