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我把内容放出来，请各位分析一下，谢谢, P7 ~: f. B) _$ F' i$ @; Q
Cell culture
. a* w7 o. \2 p) b- ROG2-MEFs were cultured on gelatin-coated dishes in normal MEF media: high-glucose D-MEM) @9 v$ T( A0 K8 U
(Invitrogen) with 10% FBS, 0.1 mM non-essential amino acids, and 2 mM L-glutamine. piPS
$ e! `4 `& L, q2 p+ h; B9 |cells were cultured on irradiated CF1 MEFs with normal mESC growth media, which consist of
7 t# W3 {; u/ w* L6 R+ @Knockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 mM 2-ME, 2 mM L-glutamine,9 G0 `! n# @/ P: ]8 O
0.1 mM non-essential amino acids, and 1000 units/ml LIF (ESGRO, Chemicon International). The L: |& B2 \4 ~+ W' K& H5 ?; E
piPS cells were passaged every 3 days as a single cell suspension using 0.05% trypsin/EDTA
3 }$ T* U' j% ]8 r5 D) Gand seeded at 1.0x104 cells per cm2 for routine culture. For feeder-free culture, cells are grown
1 z1 S0 C0 V2 j: Q" Qon gelatin-coated tissue culture dishes in chemically defined media, which consist of Knockout
5 Y/ y x1 u4 T) Y/ KDMEM supplemented with 1xN2, 1xB27, 0.1 mM 2-ME, 2 mM L-glutamine, 0.1 mM nonessential$ o+ v: ], O* r
amino acids, 50 μg/ml BSA fraction V (GIBCO), 103 units/ml LIF and 10 ng/ml BMP4
. x. ?! G! O2 y. V0 q8 w(R&D).) ?+ | v8 T: H E
Generation of piPS cells* T4 F. F! p$ I q4 z4 v5 w+ c
OG2-MEFs were seeded at 5x104 cells per well in a 6-well plate coated with gelatin in normal
6 @. [ }- J5 aMEF media (DMEM supplemented with 10% FBS). On the next day, media was changed to the
7 Q( E6 v2 T8 a7 V" s! S" T( Cprotein transduction media, which were prepared by mixing the recombinant reprogramming
3 E% g2 B' K; O! V. K/ }7 B4 }proteins at the final concentration of 8 μg/ml with regular mES cell growth medium
. U& r2 q1 N* x1 M% \1 _& csupplemented with 1000 units/ml LIF. After overnight culture in the protein transduction media,
8 w1 [2 o: x" P% k; f( @media was changed to normal mESC growth media, and cells were cultured for additional 36( B4 [/ U' _7 x
hours before repeating the same protein transduction cycle. Total four protein transduction
- F; ~8 _/ J6 tcycles were applied on the cells. After completing protein transduction, cells were then
* _3 p# }. G( i! W5 Y' Epassaged onto irradiated CF-1 MEF feeder cells at day 9 in normal mESC growth media. Media
y, m0 z1 k) E& wwere changed every 3-4 days until GFP+ colonies were observed around day 30-35. GFP+9 @! t8 n3 J1 D- w
colonies were then passaged onto new irradiated MEF feeder cells in normal mESC growth
; O, q6 `4 t! W. @. _media, and stably maintained and expanded as piPS cells. Some colonies were further selected
5 s' X. A1 _$ q7 _# [. fand expanded in the presence of pluripotin (1 μM) or PD0325901 (1 μM). |
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发表于 2012-3-25 09:33
