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Retrovirus / lentivirus infection protocol" W- v; R' x4 q/ b
D1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection)
t, P+ P# p. B: K5 S8 h% L*** Handle HEK293T cells gently to avoid detaching from dishes
: c1 |2 a' z* E1 m) P& iD2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction
! ]% x! T) K9 ]$ \2 VFor retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG
u. }4 \+ G0 k2 MFor Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG
8 _! S$ D1 R. X- a*** ~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)% M. x7 ]' @6 i3 f' S. P# ], R# r
D3: Seed target cells for infection in 6-well plate (60~80% confluency before infection)
/ k4 f6 V ^" r- DD4: Collect viral supernatant from HEK293T cells into 15ml tubes
2 ~' W9 ?6 P2 M' {5 R! m↓ spin down at 2000RPM for 3mins to remove cell debris# U, Q' q0 |* r
↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus)
* W) R+ X% C! L↓ Wash cell in 6-well plate with 1XPBS
# g; Q5 K, k) s& `. L↓ 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) 9 J1 L% T2 _$ E% n1 U" l, }
↓ Incubate the cells in 6-well plate at 37°C for ~6hrs! R5 D. ~$ e' w
↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%+ l* M8 Z) s L6 {) E
↓ Incubate at 37°C for overnight
3 i u2 }6 u: L* W*** 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.8 _ N. S6 y8 g# k
*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases.
( R6 _' u C5 g a*** The leftover of the viral supernatant can be stored at -80°C.; O" t' M0 h/ S L
D5: You may start selection with antibiotics if your cells don’t need further infection; a! X2 A. _' i
*** You need to titrate the antibiotic concentration for selection in your cells ahead
2 d2 u+ c" O7 }! f( v; p/ ^$ | |
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发表于 2011-11-17 08:04
