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我把内容放出来，请各位分析一下，谢谢
% k8 `, M& t5 W! D$ U; uCell culture0 ]& A' N! x2 H! P1 M) b
OG2-MEFs were cultured on gelatin-coated dishes in normal MEF media: high-glucose D-MEM5 |- K( b1 E/ }" ]8 \, A
(Invitrogen) with 10% FBS, 0.1 mM non-essential amino acids, and 2 mM L-glutamine. piPS: g) N+ K. A* k4 o% ? C+ |
cells were cultured on irradiated CF1 MEFs with normal mESC growth media, which consist of
( r) A0 S8 b; B2 |: k8 z' ZKnockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 mM 2-ME, 2 mM L-glutamine,
# J) J3 s+ a6 u" a" g- S5 w8 Z0.1 mM non-essential amino acids, and 1000 units/ml LIF (ESGRO, Chemicon International). The! T( \7 e- n! k4 v* w
piPS cells were passaged every 3 days as a single cell suspension using 0.05% trypsin/EDTA
- S n* `1 T2 ]7 Hand seeded at 1.0x104 cells per cm2 for routine culture. For feeder-free culture, cells are grown' o8 c( s$ {. J
on gelatin-coated tissue culture dishes in chemically defined media, which consist of Knockout
5 W" `# D! E; |5 D" uDMEM supplemented with 1xN2, 1xB27, 0.1 mM 2-ME, 2 mM L-glutamine, 0.1 mM nonessential
) \3 l% M- i! x8 |: zamino acids, 50 μg/ml BSA fraction V (GIBCO), 103 units/ml LIF and 10 ng/ml BMP4
6 M, k0 P( r4 _) h- H2 F. l$ D(R&D).1 D7 z, Y/ A( x# H& S5 A. [& l8 D6 e
Generation of piPS cells6 {4 k, v" x; w& @) B
OG2-MEFs were seeded at 5x104 cells per well in a 6-well plate coated with gelatin in normal
( Q# F% G8 e4 ]0 N% N+ C: \MEF media (DMEM supplemented with 10% FBS). On the next day, media was changed to the
1 w: x4 ?- {7 hprotein transduction media, which were prepared by mixing the recombinant reprogramming
. M, ^, P% J- E& x. G' X. ~proteins at the final concentration of 8 μg/ml with regular mES cell growth medium" J& O4 k( f- L3 V j9 _
supplemented with 1000 units/ml LIF. After overnight culture in the protein transduction media,( U9 _$ `$ E+ l+ M% A- p
media was changed to normal mESC growth media, and cells were cultured for additional 36 L1 H$ ]3 ]2 T4 \2 d/ z
hours before repeating the same protein transduction cycle. Total four protein transduction$ s5 H$ P1 A" x+ c( H2 j0 { \: S
cycles were applied on the cells. After completing protein transduction, cells were then
# i, J! J5 K3 y5 w% |passaged onto irradiated CF-1 MEF feeder cells at day 9 in normal mESC growth media. Media/ ]! N$ Y0 e f, w3 e3 A/ ^8 F
were changed every 3-4 days until GFP+ colonies were observed around day 30-35. GFP+. k; L8 L2 K. u- V& B
colonies were then passaged onto new irradiated MEF feeder cells in normal mESC growth9 n2 x- s7 x& E
media, and stably maintained and expanded as piPS cells. Some colonies were further selected
; N7 n2 f6 E0 Z$ v& c4 Rand expanded in the presence of pluripotin (1 μM) or PD0325901 (1 μM). |
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发表于 2012-3-25 09:33
