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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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: S$ I# ?; y& |$ s/ X, l! s本文系xyzengh版主原创 非常感谢! m' k4 t% K$ t% e+ p* |1 e6 U
* ]/ k( ~4 b. ?/ z6 m6 L- ^& xIPSC Generation by Lentiviral System Protocol
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- y4 o$ x# Q% Z( P: YLentiviral Packaging
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7 _; \- a$ X' P8 DMEDIUM' g2 K2 u" x6 E& T
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
" [0 c! `/ V0 Z/ S- K$ y. k" Y293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.& r7 `- G H3 F5 D3 Z; {5 j
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293FT CELL CULTURE& o' n; Z' r3 i: C# S" [+ ]
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 9 s8 t- n0 h+ t+ E$ q4 W# P
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REPROGRAMMING FACTORS
3 z% T2 Z4 D- T7 U1 k5 hOct4,Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)5 r, E4 w" S7 {8 o
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Materials Each T75 flask) }$ d+ W7 `# I5 B5 X5 c' w
293FT cells 10-15x106
. }$ L7 Y0 m+ Y. D' Y. W3 b+ jMD.G (VSVG) 5 µg
2 Z. w y( @- VPsPAX2 10 µg% J* V! P" U0 O
PSIN vector (~10kb) 5 µg
$ U" L6 t5 N3 q/ ~Superfect (Qiagen) 40 µl
+ F! ~, {8 j6 |2 S+ YIMDM 400 µl! \7 o4 n/ m9 Q& \9 z
293FT medium 10ml
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/ N& ?/ Y: V8 y1. Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
, L, O; {) @: Q2. Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
8 }! ?# G- f4 R+ U* t3. Add 10 ml of 293FT cell suspension to each T75 flask.
& O3 m- P4 u2 l( m: k4. Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
$ j. A' o8 U _, g! x% i5. 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.
5 ~5 }1 l( J9 D h4 _6. Incubate the DNA/superfect mixture at R.T. for 10 min.
/ [# D; x3 [$ a3 f# {, Y; T7. Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
, ]1 r$ V6 p2 @3 l" f- B8 \8. Incubate the cells at 37ºC O/N.- u1 ]% Z' O8 e* [2 M& ?* j
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.! @1 q% `$ F: K' r: w) `% S/ k. x
10. Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.4 E! R2 A7 g" N$ n1 g* Y+ A
11. Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
+ V- | ?( r$ m) g8 s; o7 c12. Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
@3 C9 }1 v9 O13. 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.- ^6 Q* ~) l+ n4 k6 j' w0 W7 {
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Preparation of human fibroblast cells (IMR-90)3 _/ s9 z y+ |, }
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.
/ g% t' U% k+ q3 [ J7 A2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
6 d* F9 U) X2 j0 @3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant. ~# X1 A; b* _- t
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.; S0 N. t1 {, m( {) v! M6 U
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.$ s( T. {+ u' i9 o& b9 C
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
7 }6 U% [" D+ `4 Z; E& ?7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection0 g' s$ F4 ?3 q6 Y1 ?) M
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.: j9 Y0 X: [) a9 H. j
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.8 |6 d( R" w- |5 Q2 i2 C: x& D* c
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.
1 d6 ~1 [) T+ }. a: g4. Repeat transduction (including virus harvest) as described above.* @6 ^" s( c' \/ T- c
5. A 3rd transduction may be necessary.
4 p0 I7 Y, Z: m# i9 X! e/ M6. 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).
, Y( R- J; h8 F$ j$ t4 B7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
. {, ^5 _9 ~+ R2 {- M# e& }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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