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Retrovirus / lentivirus infection protocol7 j# O3 A- h, U& I( d# h
D1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection) 0 }7 w) ^8 H1 E. S) N! b! E
*** Handle HEK293T cells gently to avoid detaching from dishes / B/ f) ]$ ~) x. q4 r g+ ]
D2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction$ M( y! {/ u. W9 M4 j% A: w& t- k
For retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG
' b9 Y4 C6 u: J4 Z# z8 bFor Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG
' N: s0 w( [& a" t5 q! ]4 s*** ~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)
D1 u }! u) V3 i+ q# q0 yD3: Seed target cells for infection in 6-well plate (60~80% confluency before infection); t' i" K; J- l; s% F4 ?0 B! ~
D4: Collect viral supernatant from HEK293T cells into 15ml tubes
5 ]/ T. \4 ^3 y9 g) i7 Q% X↓ spin down at 2000RPM for 3mins to remove cell debris, X, Q* c- k k: W2 `& U" y
↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus); Y, w6 G( [! O6 y5 Z9 j
↓ Wash cell in 6-well plate with 1XPBS
3 V! X% l0 V" d↓ 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) * B' S& F% z( O* ^6 Q( t
↓ Incubate the cells in 6-well plate at 37°C for ~6hrs i% Q& y) l$ Y
↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%
4 Q* U8 N% `! V3 r( x" t$ O- r↓ Incubate at 37°C for overnight
& A) m0 y; I x*** 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 Y8 ^. T5 a( A( |8 B
*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases.
" q2 g7 j6 `0 _1 q*** The leftover of the viral supernatant can be stored at -80°C.- [$ `0 Q' B+ b
D5: You may start selection with antibiotics if your cells don’t need further infection
8 Q; f0 V9 m8 X5 v( t8 C*** 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
