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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
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IPSC Generation by Lentiviral System Protocol
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, e5 ]; m( M5 R J7 o3 `Lentiviral Packaging
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4 p* f% P6 p: }1 }MEDIUM) V9 r, D( q/ U7 i( @! ]
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
! H5 E% W- N, b, { W8 y9 G7 T293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.# q4 x9 F0 \0 T; @
# `# \/ ]% {3 D8 v2 Y7 \293FT CELL CULTURE
' R4 s1 a7 g/ r" Y2 jMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. ) S- j9 V& D0 ^5 P& ^& w6 h$ V/ Q
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REPROGRAMMING FACTORS: Z6 q! H' f0 R/ `+ F4 Q+ M
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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$ ~# P* E! O- r( R! k- n d7 H9 L8 ALENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask
8 _; _# m& ], t: t293FT cells 10-15x106
' N( ~- j( X" G5 xMD.G (VSVG) 5 µg
$ w y5 |! q4 g! ~PsPAX2 10 µg
A( Q; g# l! x0 S7 {* OPSIN vector (~10kb) 5 µg! |7 i2 U' N( K% j. \7 T4 V2 f# Z
Superfect (Qiagen) 40 µl
/ ~$ x% u6 o* ^2 cIMDM 400 µl2 E! i# _- A g2 w1 n% y
293FT medium 10ml$ _: N' v) n o& e9 {1 _1 {. W* d
: l/ F* [; R/ G, j+ x1. Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
, X$ |5 s( t* l5 g _ ~2. Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).6 P' q2 C7 B7 J2 F, L
3. Add 10 ml of 293FT cell suspension to each T75 flask.& `/ F! y: f$ W6 H, ^7 z
4. Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
* Q5 A% u8 d& g- n5. 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.
7 L" @" e& s! \3 d9 U6. Incubate the DNA/superfect mixture at R.T. for 10 min.
* t0 \9 V. Y! [! n7. Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
; B8 @4 E0 t, W8. Incubate the cells at 37ºC O/N.4 i6 T! V" X% v! D; 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.) R: Z9 p' U1 n
10. Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
2 ^- A/ J8 v2 Z+ l, ]2 G e7 J11. Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
, l, |( j, n- R5 g1 a12. Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
* d/ ]+ K0 H G13. 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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Preparation of human fibroblast cells (IMR-90)
' u! i/ |# T( b2 Y1. 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.
; J, q' B. F& z2 [2 \( V5 h2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.1 g0 z1 ^- I* b7 {: {
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
' g! z+ S1 O+ y W4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.8 {$ \) e5 ]3 z- \/ v
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml./ U; m, T! e% e
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.; U9 L2 p' A5 ?0 m4 U
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection1 s2 Y" Z6 a P" c! [
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.: H( V; @+ g( K
2. 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.# x. u( F: R% s& e5 C8 Q
3. 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.
6 ]0 }# E, x2 l+ T2 B4. Repeat transduction (including virus harvest) as described above.- l9 ^. b! b$ e# u1 g: t# v
5. A 3rd transduction may be necessary.! {* k* Z$ h \' L! N( t
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).
; \$ ^% K$ J! e! |3 r7 v( F j7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.+ Y# A2 F5 G# p; ^1 K5 s
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
