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- 积分
- 306
- 威望
- 306
- 包包
- 3650
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IPSC Generation by Retroviral System Protocol6 H) U" x8 F+ Y! q/ v9 `9 O
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Retroviral Packaging 3 ~/ V$ m# }$ r9 g4 k
* \, P/ ]' O( ?5 H9 ^MEDIUM( a+ Z& t% g. s& V+ S
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
6 K( C1 a6 ~. O# ^- x293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE1 k/ D$ n; h8 v4 D1 o
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. ( b1 b# B# `$ S2 M5 z/ P! {" q+ ^
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REPROGRAMMING FACTORS1 [3 `; e4 p9 j, e. W2 L8 I: {
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4/ }$ a& m0 k$ b' x2 k# m+ u; Z! Q! N
% W* ~) ?% O4 y" Y4 I5 @Transfection of 293 FT Cell with Lipofectamine 2000
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For T-75 flask( j6 ~8 q+ o1 ~) ?/ f
Prepare 293 FT cell:
+ z* Y5 U- w) T5 rPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
8 f6 o) N' l& u, D- XObserve cell dish before transfection. The density of the cell should be 80-90% confluent, and the cells should be evenly distributed and attached on the dish.
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. 6 r7 Q, y' U) I. M& ^
2. For each T-75 flask, add 40 ul Lipofectamine 2000 transfection reagent, 600 ul of IMDM, and mix; incubate at room temperature for 5 min. # ? T( j3 V. H; p4 Q+ Y2 b
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
) c4 R2 L! M+ _- U4. Mix and incubate at room temperature for 15 min.4 l* b/ b- v# u" O8 |
5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.1 Y. {* |% g6 n& P# L5 C* Q! @
6. Incubate at 37 ºC, 5% CO2 for 48 h.1 x, T0 b! L3 g2 \$ K: R5 Y
7. Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.. L; n- o, I7 Q% ~
8. Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
b5 R$ R3 ]: S) _ N9. Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). : Q- ^# `0 `2 A$ H/ Y' Y* w$ G$ D8 a
10. Use filtered virus directly or store in 600 ul aliquots in cryovials at -80°C until use. Retrovirus can be stored at -80°C for several months without loss lot of infectivity.
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- G' N& Q* r% KPreparation of human fibroblast cells (IMR-90)
! ]3 K7 r, B; J& f3 x; F" I1. Day 3 (~ 10am): when fibroblast cells have reached 80% confluence, aspirate medium, wash once with PBS, cover cells with 0.05% trypsin, incubate for 5 min at 37 ºC.
6 m3 ?/ D- \0 z2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
% R- T% h$ p% q2 A3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.* s7 ?" C8 l3 z L
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer." T! B% `7 d* m& x2 e+ U7 ~2 ^
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.0 \+ G9 D7 d5 l3 o- w& v$ B) }7 J
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
, k8 s( d* {# m0 A0 m- t# y7. Incubate at 37 ºC, 5% CO2, for 6 h.. w/ w6 C1 r6 s2 D3 L9 J0 C
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Retroviral infection3 C6 }) d% x* r* v
1. Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.* b6 k5 A. J1 g1 G. Q$ v" x
2. Day 3 (~ 6pm) Add equal volume (600ul) supernatant of each factor ( OCT3/4, SOX2, c-MYC, KLF4) to fibroblast dish in the presence of 10ug/ml polybrene, incubate at 37 ºC, 5% CO2, for 2h.+ i, [' `0 Z# B/ I/ z# X& \5 o
3. Day 3 (~ 8pm) Add another 2ml fresh fibroblast medium and incubate at 37 ºC, 5% CO2 for overnight, then aspirate the virus medium and replace with 2ml fresh fibroblast medium.$ e1 T9 K6 g2 ]' @! r
4. Repeat transduction (including virus harvest) as described above.
/ @, z, `5 ^) m5. A 3rd transduction may be necessary.
) b: t5 b2 h+ q/ r6. Day 7 (9~10am) Trypsinized fibroblast cells completely (4 days after 1st transduction), transfer to two 100-mm MEF dishes (3 x 10^6 MEF cells /dish)., T4 y; N7 \& K, t h' k5 b
7. Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
+ L4 P) d: x- |" ?8 `! \) p8. Day 17 (9~10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up. |
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发表于 2010-5-5 23:10
发表于 2010-5-5 23:34
发表于 2010-5-6 07:25
