逆转录病毒的包装

zhangtang

1.我有一个含目的基因的逆转录病毒质粒，想将该质粒包装成病毒后再感染细胞。由于之前从未做过病毒包装，我想请教一下群里的各位朋友，包装病毒的过程以及测病毒滴度难不难啊？是自己做好呢还是交给公司做？ % G' P3 k% F$ i9 Y' a' A- d
2.如果这个逆转录病毒质粒不包装成病毒，可以直接拿来转染细胞嘛？

momocy

包装病毒并不难，一般用293T或293FT做转染，生产病毒，再感染目的细胞，我给你上传一份慢病毒和逆转录病毒的protocol，你看看，很简单。测滴度也不难，病毒感染293T细胞后的48h测，步骤如下： ①. 15万293T细胞48h即可刚好长至90-100％满，此时即可固定细胞，计数确定病毒滴度。
0 Y1 N1 u3 e1 @9 A& U ②. 将培养基用真空泵吸去部分，务必不要将其吸净，由于293T细胞易飘，固定及DAPI染色整个过程请务必保持293T细胞处于液面之下。用PBS涮3次。加入4％ PFA室温固定30min, 24孔板每孔200ul。5 s9 {' q; C/ M+ M
③．用PBT洗2次，每次3分钟。& J" `2 [# e9 k+ T+ C6 T, l
④．PBS将DAPI 1000倍稀释，每孔加入200ul，避光室温静置5min。
' J9 N8 u) X/ s2 |# Y2 K8 b⑤．PBS洗2次，每次5分钟，即可在荧光显微镜下计数，取2-3处取平均值。& u, G! S. ?% J
⑥．病毒滴度（IU/mL）=荧光细胞占总细胞数的百分比÷加入的病毒上清体积×1.5×10*5×10*3

momocy

Retrovirus / lentivirus infection protocol, F% w$ _. T& G) X8 v
D1: Seed HEK293T cells in 10cm dish (cell density should be ~80% confluency right before transfection) ( K0 M; V4 |2 q# p9 D9 d* H8 U
*** Handle HEK293T cells gently to avoid detaching from dishes
8 G8 b4 {- M1 e4 LD2: After O/N incubation, transfect the following vectors with proper reagents (Fugene 6, Lipofectamine 2000, polyjet et al) following their instruction" G1 D" O! t) a
For retrovirus: 12ug of gene of interest, 6ug of Gag/pol, 1.5ug of VSVG " o- W& j  o9 m+ u0 K' `
For Lentivirus: 12ug of gene of interest, 6ug of Δ8.9, 1.5ug of VSVG
% D# G4 f  B  X- 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)
9 j' e$ ~+ F* aD3: Seed target cells for infection in 6-well plate (60~80% confluency before infection)
) _' K4 b  A% S1 j( g( d: WD4: Collect viral supernatant from HEK293T cells into 15ml tubes& G. Z; a" k4 T* G2 i
↓ spin down at 2000RPM for 3mins to remove cell debris
( u3 }/ x2 D& g8 N7 G↓ pass the viral supernatant through 0.2um or 0.44um filter (called D1 virus)
& K) b8 g6 ~2 s& G↓ Wash cell in 6-well plate with 1XPBS
! D& s& N5 j* B↓ 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) 3 r( C: B9 R% ]7 l
↓ Incubate the cells in 6-well plate at 37°C for ~6hrs
7 B& ~3 P  Y- f9 ?8 b' J↓Add FBS or complete cell culture medium to make final FBS concentration close to ~10%
/ k! [( ~+ ~2 n: }, V( ?+ _( h↓ Incubate at 37°C for overnight
' h5 t3 L6 O- Z*** 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.7 T1 H# D8 N' L0 R( M- ?
*** For retrovirus, you may need to repeat the infection for three times; for lentivirus, one time infection is enough in most of the cases.* @) a* p7 {8 g  e3 B$ Q
*** The leftover of the viral supernatant can be stored at -80°C.
& U2 T9 v8 u: mD5: You may start selection with antibiotics if your cells don’t need further infection
$ P% {/ s. N6 \/ X1 c+ W! ]*** You need to titrate the antibiotic concentration for selection in your cells ahead1 i7 `3 ^1 [: O4 l7 W4 S, N

zxcv12345

回复 zhangtang 的帖子
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1如果有齐全的质粒，自己做病毒肯定没问题。2 w# H$ ^4 J) ]+ @8 \" [7 B: R
2，质粒本身就可以用来转染，效率没有病毒高

qingtinglueshui

回复 momocy 的帖子9 Z2 m& D) n) g6 J
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你好，谢谢你给我的回答。我这个逆转录病毒质粒不含荧光，那如何在荧光镜下计数来测病毒滴度呀？

zhangtang

回复 momocy 的帖子
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如果逆转录病毒质粒不含荧光，如何在荧光镜下计数来测病毒滴度？

momocy

回复 zhangtang 的帖子8 ^: Q) A. R! G5 q. [
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你增加一个GFP的对照，通过GFP的来估算其他质粒

zhangtang

回复 momocy 的帖子
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你好，你说增加一个GFP对照的话，是不是空白质粒flag也要包装啊？还GFP组就可以作为空白质粒对照组？

momocy

回复 zhangtang 的帖子6 ~+ [0 }4 i% L

9 y7 q* T0 F. F你转染的时候，多转染一个GFP，也就是ＧＦＰ和质粒同时做，算出ＧＦＰ的滴度，然后你质粒的病毒和ＧＦP加的量一样多,这样不就行了么

99牵牵

我的就是这个样子的，本身质粒没荧光，加一个GFP作对照！可是问题是我的包装细胞状态总是不好，包出来的病毒去感染293t荧光很不亮 而且很少 可以告诉我怎么个情况吗？谢谢啦!! T' Q/ G) u* U4 b5 ]