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本帖最后由 细胞海洋 于 2010-5-5 23:12 编辑 6 {' N& f/ l7 l/ c! M' \
% F. v, C7 H3 |/ t( \& e本文系xyzengh版主原创 非常感谢+ W2 x( y; n g: |& T8 T) D
$ w+ Y) L; |" Y6 [" q1 jIPSC Generation by Lentiviral System Protocol
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, p. ?, X( S. |* g# LLentiviral Packaging
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( b# g% g( n- |% |' a- XMEDIUM5 F5 R+ V. b" _# G5 `# l
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
" F9 M, ?3 B) o, U) a, Z2 [8 G8 ~293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE& i! E# M! V$ x) y3 R5 @
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. a( Q# w5 T" V* @4 b! p' T) |2 L' Y
( N0 @8 ?, s! C7 o; l# }REPROGRAMMING FACTORS
5 I# D. i" d7 W2 [* `" S% y" FOct4,Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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1 T& s* [3 U' W- D6 o7 K7 s( sMaterials Each T75 flask
2 L( y7 |) _. b# \293FT cells 10-15x106
9 x: H0 [0 ?1 W! `3 W RMD.G (VSVG) 5 µg' S# ~. @, Z! E) s; J
PsPAX2 10 µg3 E! A, ~2 X/ a j2 G- m0 q3 U
PSIN vector (~10kb) 5 µg' s' I* `& K" Y% @1 _
Superfect (Qiagen) 40 µl
1 D* L* N( J* x, x7 i/ x9 @- ^IMDM 400 µl
2 U( i u! [3 s6 L9 r: |' \- D293FT medium 10ml, T' G0 R% d0 l- ~
6 N, x9 L6 G2 \3 c1. Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.5 F6 X! F8 Q/ Y5 L( B. d3 Y! p
2. Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).( z9 P* L& y+ X U$ W5 o- X
3. Add 10 ml of 293FT cell suspension to each T75 flask.
: M2 ]. b( s" M( Q, |) L( K4. Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).3 f# I# R& V! w; D1 u7 \% \' P0 p
5. 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.) _, l5 t- `9 y
6. Incubate the DNA/superfect mixture at R.T. for 10 min.9 W6 n% P; J3 ^! _- h$ e! i
7. Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix., w$ x9 M( U) S: T( \
8. Incubate the cells at 37ºC O/N.* z( a6 P6 i) V: M% f+ p5 h
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.
% u" n; p( N0 m' K- r2 H6 B+ M10. Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
/ r2 B! W8 `+ ]" b' `11. Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.$ j" l% @' u+ E" H' W/ p2 u" Z( Q
12. Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
3 b9 k% z/ i4 f' ~0 q13. 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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, c% s% U! F& n, z+ p- g% ~3 K- @% UPreparation of human fibroblast cells (IMR-90)- X0 ^" J& S0 p3 B! z
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.
2 \2 r6 l W) D5 @' l2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
|0 D3 w* F- U/ S t3 ~3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.$ b3 O, X+ g( j) Z" x2 X1 S
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
6 b9 G3 n1 Z) v% K0 W5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
3 n, c3 L" C+ ^2 R/ `: [2 N* h, c8 C6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
9 j* l/ N' |' @7 J! s$ j5 W0 I7. Incubate at 37 ºC, 5% CO2, for 6 h.* a- ?/ {7 _$ c
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Lentiviral infection7 U% F! ]6 D' e: U$ ?5 d3 _
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
: p# K; `" Q3 {0 i% @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 R8 u0 |( W; w8 S. v- B3. 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.
# o1 V5 q# ?/ A! H4. Repeat transduction (including virus harvest) as described above.
, ?) ?" w4 a& o# ~/ O7 w% f5. A 3rd transduction may be necessary.3 e9 H/ _( T- ~/ y/ n
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).
% r% x: V! ?0 [; S. G: ?7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
c8 t& Y% e7 U2 L& e4 k4 b7 ?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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