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IPSC Generation by Retroviral System Protocol# @: v: E4 k, `! A2 x v1 y
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Retroviral Packaging " m' f% M( u9 `5 v$ T+ [
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MEDIUM
7 _: c5 P$ I9 k% K( D/ P293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.1 t! q6 L @4 S
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.0 m% G# g. m H4 L1 K" C9 } t
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293FT CELL CULTURE
2 M, \- g6 e) TMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 7 i# t7 Z' |6 {
6 ~: j( P+ f8 u; `REPROGRAMMING FACTORS
1 G# i% a8 @* N6 YpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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7 _, l7 s3 p8 ]* |2 {5 w* bTransfection of 293 FT Cell with Lipofectamine 2000
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For T-75 flask, `) I. L! f0 C3 w
Prepare 293 FT cell:9 r r2 c' Z$ d1 N, l$ U6 I+ r) ]
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.5 N+ _5 w+ S- a0 }# \6 L; o# {
Observe 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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' ]3 P0 ]/ R( H, f1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. 5 o1 h. m4 O' a2 V2 X5 F
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.
( p$ t" g4 z9 Q. B% L# J1 y5 P/ [3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
: K) _# e1 [! i% g! {+ U! Z4 G* p0 a1 X" h4. Mix and incubate at room temperature for 15 min.$ f' y- N; B7 [/ b
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." j+ G6 w1 b+ r' {! _$ {! h/ P
6. Incubate at 37 ºC, 5% CO2 for 48 h.
8 H7 n2 t; U+ h6 B. A7. Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
( |! e/ {) W2 O- n6 z; J8. Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
2 P& m& S% b( Y( P2 t# v% v9. Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
2 F7 N. p+ N9 w0 O5 @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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Preparation of human fibroblast cells (IMR-90)+ q# [0 M+ S+ O& M# s# U5 S
1. 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.; V$ c/ p$ P! i# K0 L& v' a
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.& ~: i( u* U* _! C+ o1 u/ C: s
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.: Z1 i4 h' Z% t, S
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.( O4 }% t4 k2 A0 C5 N5 k
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.. J+ s3 v: ` L" ]6 \ L
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
# E) Z* p/ r4 y0 E: [, d$ S7. Incubate at 37 ºC, 5% CO2, for 6 h.! n# r: ]: u: z# I; x
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Retroviral infection2 F+ r* g9 ^# D# v( Y! d6 @$ Y
1. Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction." z! F5 K4 w- z, F7 `! n
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.
8 o; r5 U1 q5 h+ D; m9 C4 S5 r1 `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., H! ~* p& u; j" y& u N
4. Repeat transduction (including virus harvest) as described above.; y- D* \# N6 g1 l) F
5. A 3rd transduction may be necessary.: d; h; c, y9 h6 I: B" l) j a# c
6. 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).
" M/ m n/ d3 G' d8 H7. Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
0 y5 s* r, p+ v) b8. 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
