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Protocol for Production of Lentiviral Vectors in 293T cells(pdf)( D- X9 a' T5 i. G, l
' Z: h% `" E# l' S/ zDay 1 Plating (9-10am)/ w2 v6 L& c8 x2 S) r+ y) k! F
0 ~6 h$ ~% }) t8 x' IPlate 2-2.5x106 of 293T cells per 10cm plate
" B' k8 V; Z3 [5 D& DDay 2 Transfection (9-10am)
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& l, \ _$ [0 O, r APrepare calcium-phosphate precipitate (1ml/10cm plate)
1 Q7 _1 c8 h* J C' uTransfer vector - 20µg
( |8 b2 T' h% ^$ X9 LPackaging plasmid - 15µg (3rd generation: pMDL g/p RRE - 10µg + pRSV-Rev - 5µg) 4 F+ R2 f. N$ W8 g# ~8 J2 \
Envelope plasmid - 6µg8 h7 P+ g4 O0 |" s; K) a: B
Add water to 0.5ml, add 0.5ml 2xHBS and mix well. Add 50µl 2.5M CaCl2 and shake briefly, keep in RT for 20-25min, add dropwise on a plate and mix gently with a medium
3 O3 g, ^% p7 c* |7 U1 D, ~Change medium (6-8hrs later); remove medium with precipitate and add 6ml/plate of fresh medium
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" w5 t/ h3 g ?+ {/ yDay 4 Collection (9-10am)/ b, _0 {. S2 `4 e6 B6 s# |1 D' y
' h4 @, N% h3 _# ~9 N5 s
Collect medium ' R. z% r$ r; q1 Q9 y! K
Spin 3000rpm/5min/RT
1 Q7 O5 C0 a. t; n# z* YFilter through 0.45 µm# g- s' v4 N2 R9 s6 y! k
At this point virus can be used for transduction, frozen at -70°C for future use, or concentrated
7 Z1 S8 j* o' t- B M. a) a$ FConcentration
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+ K2 B! Y: L; H$ r" q/ Z! `- Y8 tTransfer 30ml of virus to 33ml Beckman conical tubes spin at 26.000rpm/2hrs/4°C in Beckman SW28 swingle bucket rotor. After spin discard supernatant and resuspend the virus in a desired volume of serum-free medium (e.g. Cellgro or Episerf) or PBS/1% BSA, aliquot and store at -70°C. For transduction of very delicate cells the virus can be concentrated on sucrose cushion, just put 4ml of 20% sucrose on the bottom of the tube and overlay with 26ml of viral supernatant.
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Reagents: 7 H& _1 ]$ L, K
$ [& d$ y/ M3 _/ w. D4 `2 x HBS (for 500ml)
8 F! s9 K( t) i2 hNaCl - 8g
3 M+ \4 u% b5 P* A' p) t! GKCl - 0.38g
% J$ R. Y4 g" \; e& nNa2HPO4 - 0.1g
/ O5 R, W# k* a- S$ y- [) VHepes - 5g 2 L; T- ~0 c/ i4 U2 S% o
Glucose - 1g Bring pH to 7.05
: e( h" C; g6 @6 j0 B% @0 i8 Y$ X2.5M CaCl2( A" y% x6 ]5 U: O
bi-distilled water |
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发表于 2011-2-21 11:23
