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Retrovirus / lentivirus infection protocol7 ?; m6 Y1 N8 e7 q1 W5 |1 v
D1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection) ) T5 E4 n7 {3 M4 O1 s$ n3 {
*** Handle HEK293T cells gently to avoid detaching from dishes
/ d; D9 m2 K7 S& `' k& @8 @D2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction8 g1 k/ f0 K% w) p4 J8 }
For retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG
, B& ?: ~3 p, L/ K* Q( {For Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG " Z8 J! D( l# u" D
*** ~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)3 `; |5 b$ x* d7 t3 v
D3: Seed target cells for infection in 6-well plate (60~80% confluency before infection)" |$ C7 z* ^# x" a4 L, U
D4: Collect viral supernatant from HEK293T cells into 15ml tubes5 K6 r: E, ^7 t% R; A D7 S2 J
↓ spin down at 2000RPM for 3mins to remove cell debris) Z5 D# t3 ]- g2 J. F
↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus)3 k7 Z" U; I3 t' y3 @
↓ Wash cell in 6-well plate with 1XPBS
e- }9 o, g9 l- N j6 a' {↓ 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) " c5 z: B/ U; C7 |. {9 @- k
↓ Incubate the cells in 6-well plate at 37°C for ~6hrs5 t, `4 J5 v% i% O
↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%
4 Z! r* [. Y& j) W5 u* L↓ Incubate at 37°C for overnight. A2 w, }0 L) o9 x; F! G7 ~
*** 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. \$ i$ h, n9 H: O
*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases." {7 ]9 o. C4 ]% h! n; \9 y1 U) O' c
*** The leftover of the viral supernatant can be stored at -80°C.) K5 d- C3 `* c" y6 u
D5: You may start selection with antibiotics if your cells don’t need further infection
( X& D& h( n- L*** You need to titrate the antibiotic concentration for selection in your cells ahead% W$ }! J) M9 |' Z9 a% R5 U
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发表于 2011-11-17 08:04
