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Retrovirus / lentivirus infection protocol x) l8 B. Y/ q$ i
D1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection) ( E6 F/ C& o# m
*** Handle HEK293T cells gently to avoid detaching from dishes 8 s5 q6 e* l. A0 w: i. b/ a4 K+ d8 O
D2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction; c3 F6 [4 V0 L/ Z6 h- s |7 O; X R
For retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG 7 M M: y" Z3 M
For Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG ! x) I3 {9 ?4 T
*** ~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)
1 u. Q: z' r6 o$ J! `; CD3: Seed target cells for infection in 6-well plate (60~80% confluency before infection)
( l& {/ k9 c8 [9 o; yD4: Collect viral supernatant from HEK293T cells into 15ml tubes3 f" q+ a6 p; v- r) ^: i/ J' ]
↓ spin down at 2000RPM for 3mins to remove cell debris
( i8 W- Y0 x" H p↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus)
a3 `: T, ?$ x5 ]; a↓ Wash cell in 6-well plate with 1XPBS
M a; Y- k- ?- w* p6 H# H" ?0 B7 q↓ 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) ; e+ B! _1 Z, r7 }% c7 [% c5 K
↓ Incubate the cells in 6-well plate at 37°C for ~6hrs/ ~5 Z; S2 l: D( {0 @& Q
↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%
9 g2 F1 ^) d6 u! O↓ Incubate at 37°C for overnight" o4 a$ W# ?3 ` X/ F6 X5 T
*** 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 Q7 T( a/ q* a( F" C
*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases.1 N" f' Y# H i& M# G8 j4 x; F
*** The leftover of the viral supernatant can be stored at -80°C.0 z0 ~; z s; S- {: i
D5: You may start selection with antibiotics if your cells don’t need further infection
6 X+ k) B% ^1 q$ N/ B4 w9 c: |*** You need to titrate the antibiotic concentration for selection in your cells ahead. R* r7 Y1 K- ~3 T; }& w7 X
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发表于 2011-11-17 08:04
