|
 
- 积分
- 348
- 威望
- 348
- 包包
- 1396
|
Retrovirus / lentivirus infection protocol0 V! w I3 `: }( q5 Y' J% c. ~7 Z
D1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection)
4 P( D0 n8 H( a9 n*** Handle HEK293T cells gently to avoid detaching from dishes
4 J7 n: g' h0 b' q# D1 t! i0 ^D2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction
- G6 m) ^) p( w% n b f$ NFor retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG
& X8 f9 w2 m( V6 K3 Z( ]' B2 ]5 EFor Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG ! @$ q, X( f3 j0 X
*** ~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 Q! T i9 F1 n0 a
D3: Seed target cells for infection in 6-well plate (60~80% confluency before infection)# Q+ V" }) E5 ^, d, @8 {) g; c; N
D4: Collect viral supernatant from HEK293T cells into 15ml tubes
0 M! Z* Y" b7 D: R↓ spin down at 2000RPM for 3mins to remove cell debris
+ \! U. e3 P2 ~2 B; Q) ^4 `+ X↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus)1 R( k8 n' N! `7 @5 g
↓ Wash cell in 6-well plate with 1XPBS3 ^" t7 O+ S: c: C4 S0 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) ) L# A4 F2 s. \7 w9 l0 F9 V
↓ Incubate the cells in 6-well plate at 37°C for ~6hrs6 x! M1 I8 f) P2 v( K/ t
↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%
0 [4 r6 f* |, Q& F) z3 ]% m* ?7 x; p↓ Incubate at 37°C for overnight; I0 E% F$ x2 ^6 _
*** 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.
; c0 A c9 l, ^# v3 a2 K% E$ a*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases.; p3 ~) l7 z+ \4 t, T; X: m
*** The leftover of the viral supernatant can be stored at -80°C.
: h2 n+ C* D: E* [' |$ o8 O. QD5: You may start selection with antibiotics if your cells don’t need further infection) m* @5 r, X1 ~1 `7 _% a
*** You need to titrate the antibiotic concentration for selection in your cells ahead
3 B# Q% P: z5 l5 Q3 b! q9 ^! P |
-
总评分: 威望 + 5
包包 + 10
查看全部评分
|
发表于 2011-11-17 08:04
