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Retrovirus / lentivirus infection protocol! f( o& e2 ]4 I" |/ D4 A! |' r) ~
D1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection)
. S! V5 z ]0 r1 }- u6 L*** Handle HEK293T cells gently to avoid detaching from dishes + r9 V) q& C$ }8 G' E' S' ^1 t0 R X5 _
D2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction
. o+ g7 `' [" H. N/ YFor retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG
9 {& F* B9 g' @+ dFor Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG
# k0 f% k) \+ P5 r5 ^*** ~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)
+ [9 g: o4 R, a8 h. N# a$ iD3: Seed target cells for infection in 6-well plate (60~80% confluency before infection)
! ?: C+ h9 A2 j [, v. F7 iD4: Collect viral supernatant from HEK293T cells into 15ml tubes
/ `0 V7 Y- |$ y5 j- o' R↓ spin down at 2000RPM for 3mins to remove cell debris
2 f; y7 @/ _( g% {+ _↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus)
4 L. e( k) f3 I↓ Wash cell in 6-well plate with 1XPBS
+ O i1 V4 {/ u+ j+ ~" `2 ?↓ 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)
6 z' V/ b2 A6 s6 \1 A↓ Incubate the cells in 6-well plate at 37°C for ~6hrs
" k0 ?9 m$ f+ c* D) i1 @8 q5 J↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%, ?! r7 `- o; j) G+ w& P
↓ Incubate at 37°C for overnight* o! F* U* r _; F* L8 f
*** 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.
2 H7 ^! T5 P( a- i, x*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases.
$ r: H& K- u1 c) o: t O/ P) s*** The leftover of the viral supernatant can be stored at -80°C./ [8 R) i0 C, c+ y6 b7 x6 o
D5: You may start selection with antibiotics if your cells don’t need further infection7 Z) {" D- A( J. @& q, V
*** You need to titrate the antibiotic concentration for selection in your cells ahead* k+ U" e& r5 A2 K8 r8 z' Y
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发表于 2011-11-17 08:04
