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本帖最后由 细胞海洋 于 2010-5-5 23:12 编辑
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本文系ｘｙｚｅｎｇｈ版主原创 非常感谢8 M$ B" i- d& m9 d* d
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IPSC Generation by Lentiviral System Protocol% [' }7 H9 I" n; a
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Lentiviral Packaging
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MEDIUM/ G. n' v. j) y M
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids. c( S- m. I2 X+ B% W
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.) y" y4 T+ |1 c9 I
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293FT CELL CULTURE& m; [' ^/ l0 l+ A
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS& r# T" S' W0 |$ n& k. B- f2 a. h
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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2 N! A. q- L3 p3 Y- ]) Q: j/ }LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)+ h7 T8 ^0 V/ ^" R5 N
7 J4 ]; u" t0 V! ~. g6 dMaterials Each T75 flask1 Q6 G9 I8 X7 s% G2 {( @
293FT cells 10-15x106
+ w" Z$ q! Q7 ?1 uMD.G (VSVG) 5 µg
/ E3 ?4 W4 i D& C1 C" LPsPAX2 10 µg: O. Q M7 Q. t2 @8 g1 Z* K
PSIN vector (~10kb) 5 µg
" {( Q4 p1 P% m; `) R TSuperfect (Qiagen) 40 µl ' J F) m" ^7 r7 ]; i7 p9 o, F; O
IMDM 400 µl
& v( ?0 f% f8 B. b3 T- k1 ^293FT medium 10ml! r% W$ ]* \9 L. R2 c
! Q! S) I9 h Q; ? h' V0 {1. Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
1 R9 d8 D3 U7 Z W# s4 {4 S; h: i2. Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
" B% N; m, d- D4 ]/ `& Z7 l. z3. Add 10 ml of 293FT cell suspension to each T75 flask.
& S2 f& f, i/ V$ \3 n4. Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
( u5 j3 n2 X& t4 R5 Q5 ^% p5. Add corresponding amount of Superfect to the DNA solution, and mix with brief vortex. Briefly spin down to collect liquid at the bottom of the tube.
1 R& L, b8 k, C( N0 Q7 s; S! @6. Incubate the DNA/superfect mixture at R.T. for 10 min.
% I# A/ ]' k( A% j( k7. Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
3 W9 g; c# Z, A7 U) Y! J# U8. Incubate the cells at 37ºC O/N.. _. R1 g5 i7 \9 F3 s
9. Day 2: The next morning (9-10 am), remove the medium, and add 8 ml of 293FT complete medium (with sodium pyruvate) to each T75 flask.
5 H8 \9 {. C2 a& P" z: q f10. Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
- [: |* |% p4 t' b, v! H" W% Y7 u- @11. Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
4 T# r8 Q) M/ R/ L. F, h6 y6 P6 _12. Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). " t( o5 }/ F4 F8 P9 d
13. Use filtered virus directly or store in 800 ul aliquots in cryovials at -80°C until use. Lentivirus can be stored at -80°C for several months without loss lot of infectivity.
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7 g: D4 A2 z% W: S' @7 q! LPreparation of human fibroblast cells (IMR-90)2 }7 \9 `0 B& J2 m$ e
1. Day 4 (~ 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! q# h5 Y: }" ^& R7 t5 @$ m) J8 A4 [2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
6 X& M! P" \, h2 Y3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.0 W1 d4 D2 d2 V( b
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
5 t5 \) E' H) B) ] I, |5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
5 [1 C2 P; p. n# u6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
) J. V# i) }9 o6 F9 n3 _7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection
* A6 O( p4 l# S$ p- d1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
- d N+ b b j/ }5 F2. Day 4 (~ 6pm) Add equal volume (800ul) supernatant of each factor ( Yu1, Yu2, Yu3) to fibroblast dish in the presence of 10ug/ml polybrene, incubate at 37 ºC, 5% CO2, for 2h.
5 |4 g8 r& Z& n5 z( [+ h* g3. Day 4 (~ 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.8 b Y) K" U6 I' }6 ~
4. Repeat transduction (including virus harvest) as described above.0 t* y% k$ f! Y
5. A 3rd transduction may be necessary.4 ^; Y7 K4 f5 d
6. Day 8 (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).$ Z# J) u" x$ [: H/ {
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.# h. y* r6 k( f, D- ?% B* p
8. Day 18 (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 2013-1-20 13:04
