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Retrovirus / lentivirus infection protocol
/ x* n1 R8 B5 E( t) iD1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection)
$ u" F8 y* Z+ E, L G*** Handle HEK293T cells gently to avoid detaching from dishes
1 d! \9 ^4 }# ]' r0 M) ?+ ~; MD2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction
* O2 F R4 `2 tFor retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG
" Y: z/ y( K( P0 i* {For Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG % c' x/ B7 N: ?
*** ~6 hours after transfection, change medium to fresh DMEM with 1% FBS supplied with P/S, and incubate at 37°C for ~48hs (two overnights)( H& A: _" r0 A. u8 `- _" [
D3: Seed target cells for infection in 6-well plate (60~80% confluency before infection)
/ n' o( N; s% V' H. Z) XD4: Collect viral supernatant from HEK293T cells into 15ml tubes
) \0 F3 T! A; W' O! `7 J5 y& J↓ spin down at 2000RPM for 3mins to remove cell debris
" ~5 O5 S! q- U+ }↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus)2 p! W; k2 J" I! j e
↓ Wash cell in 6-well plate with 1XPBS5 [$ Z; ]/ H) h4 b- b! R# _, ` x1 J
↓ Add 1.5ml of filtered viral supernatant to target cells in 6-well plate, then add 8ug/ml of polybrene (by adding 1.5ul of 8mg/ml stock) " v. T- m8 z# y- h
↓ Incubate the cells in 6-well plate at 37°C for ~6hrs
8 u7 t6 `$ g5 @↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%' x. q1 p/ O9 m" p7 k
↓ Incubate at 37°C for overnight! ~1 q0 L1 Z5 E" ?
*** After collecting D1 virus, you can add more DMEM with 1%FBS into HEK293T cells and collect D2 virus after incubating overnight. But the D2 virus usually has lower viral concentration.0 n* j% T# Z4 I) M) K, X$ l
*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases.
; u9 X1 B6 P3 l$ N N ^*** The leftover of the viral supernatant can be stored at -80°C.2 c: S1 O: Y7 g5 l8 J6 n! d
D5: You may start selection with antibiotics if your cells don’t need further infection( k, ]% ?5 U) x& G( T! a. ~
*** You need to titrate the antibiotic concentration for selection in your cells ahead
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发表于 2011-11-17 08:04
