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TSB慢病毒包裝試劑盒使用說明
發布日期:2016-05-27 13:35:40 瀏覽次數 :
導讀
TSB慢病毒包裝試劑盒使用說明
 
 
  • 實驗流程
 
制備慢病毒穿梭質粒及其輔助包裝原件載體質粒,三種質粒載體分別進行高
純度無內毒素抽提,共轉染 293T 細胞,轉染后 6 h 更換為完全培養基,培養 48和 72h 后,分別收集富含慢病毒顆粒的細胞上清液,病毒上清液通過超離心濃縮病毒。
 
二、實驗材料
 
(一)慢病毒載體、包裝細胞和菌株
該病毒包裝系統為三質粒系統,組成為 pspax2, pMD2G, pLVX \ pCDH\pLKO\pTSB

  1. 表達載體信息
權陽生物所使用的慢病毒載體多為 SBI的 pLVX系列載體和 clotech的pCDH系列載體為基礎,加以改造并驗證的pTSB系列載體。

2) 包裝質粒信息如下:
   我司的慢病毒表達質粒使用2代及3代包裝系統均可,推薦一下兩種包裝體系:
第二代包裝質粒:
PSPAX2 及PSPAX2載體圖譜和序列信息
質粒配比:
packing mix:PSPAX2:PSPAX2  3:1
     包裝體系:packing mix:pTSBLV 
 
第三代包裝質粒:
PLP1,PLP2 和 pCMV -VSVG。
質粒配比:
packing mix:PLP1(3):PLP2  (2):pVSVG
     包裝體系:packing mix:pTSBLV  1:2
 
(二)細胞株293T,慢病毒的包裝細胞,為貼壁依賴型成上皮樣細胞,生長培養基為 DMEM(含 10% FBS)。貼壁細胞經培養生長增殖形成單層細胞。
 
(三)菌株大腸桿菌菌株 DH5α。用于擴增慢病毒載體和輔助包裝載體質粒。
 
三、包裝細胞 293T 細胞的培養
 
(一)293T細胞的凍存
 
隨著傳代的次數增加,293T細胞會出現生長狀態下降、突變等。為了防止
此類現象的出現,我們需要在開始就對細胞進行大量凍存,以保證實驗的穩定性和持續性。在細胞對數生長期進行凍存,增加細胞復蘇成活率。
 
  1. 去掉上清液,加入PBS洗去殘留的培養基;
 
2、加入0.25%的胰酶,消化1~2min后,鏡下觀察細胞變圓,細胞間間隙加
大時,去除胰酶,加入新鮮培養基吹打混勻,移入離心管中。
 
3、細胞計數,將細胞全部晃下,加入3mL 37 ℃ 預熱的10%DMEM,用10mL 移液管進行吹打,較大力吹打6-8 次即可,不留死角,之后,將所有細胞吸出,置于15mL 離心管中,取50ul 混勻后的細胞于1.5mL eppendorf 管中,加入450ul 10%DMEM,即為10 倍稀釋,混勻,取10ul 細胞于計數板中計數。計數板上共4 大格,每大格16 小格。計數時,4 大格均計 數,總數除以4(得每大格細胞數),再乘以10(10 倍稀釋),即 為實際n萬/mL 細胞濃度。
 
4、細胞離心,1000rpm,5min。去掉上清。
 
5、根據細胞計數結果加入細胞凍存液(70%完全培養基+20%FBS+10%DMSO),重懸細胞,密度為3 x 106個/ml。
 
6、分裝進細胞凍存管,放入凍存盒中,放入-80℃超低溫冰箱。
 
7、第二天將細胞放于液氮罐中長久保存,并作記錄。保存過程中,要不時復蘇細胞檢測細胞存活率,觀察細胞狀態等。
 
(二)293T細胞的傳代
 
當細胞生長到匯合率達到80%~90%時需要對細胞進行傳代操作,以擴大細胞數量,維持細胞良好的生長狀態。
 
  1. 消化細胞,方法同細胞凍存。
 
  1. 細胞離心結束后,加入完全培養基重懸。
 
3、根據具體情況,將細胞分到10cm培養皿中,每個培養皿補足到10ml培
養基。
 
  1. 將培養皿平穩放回37℃、5%CO2和95%相對濕度的培養箱中培養。
 
(三)293T細胞的復蘇
 
當細胞傳代次數過多,細胞狀態變差時,或者細胞出現污染事故時,需要丟
棄并對最初凍存的細胞進行復蘇。
 
  1. 設置溫度為37~42℃的水浴。
 
2、查看細胞庫記錄,根據記錄從液氮罐中取出凍存的細胞(需戴上棉手套,
防止被凍傷),迅速丟入水浴鍋中并快速晃動,盡量在1~2min內使細胞溶液完全
溶解。
 
3、將細胞溶液轉移到15ml離心管中,并在其中加上1ml新鮮的完全培
基,混勻后離心,1000rpm,5min。
 
4、去掉上清,加入5ml新鮮的完全培養基,混勻沉淀后,轉入6cm培養皿。
將培養皿平穩放入37℃、5% CO2和95%相對濕度的培養箱中培養。第二天觀察細胞存活率。給細胞換一下培養基。以后每天觀察細胞生長情況。
 
四、慢病毒包裝和濃縮
 
  • 質粒擴增
 
構建好的慢病毒載體和輔助質粒需經過大量抽提,濃度大于1ug/ul,A260/280 在 1.7-1.8 間方可用以包毒。推薦使用 Qiagen 大抽試劑盒進行質粒的大量去內毒素抽提。
 
  • 傳 293T 細胞
 
將培養 293T 細胞 T75 瓶中的培養基吸凈,加入 2mL 4 度冰箱取出的 0.25%
胰酶,使其均勻覆蓋瓶底,置于 37 度培養箱中 3-5min,取出,搖晃可發現細胞于底部脫離,將其全部晃下,加入 3mL 37 度水浴中預熱的 10% DMEM,移液槍用 10mL 移液管進行吹打,較大力吹打 6-8 次即可,不留死角,瓶口處較難吹打可將移液管對準培口,小力將培養基打出即可覆蓋到接近瓶口的細胞。之后,將所有細胞吸出,置于 15mL 離心管中,取 50ul 混勻后的細胞于 1.5mL eppendorf管中,加入 450ul 10% DMEM,即為 10 倍稀釋,混勻,取 10ul 細胞于計數板中計數。計數板上共 4 大格,每大格 16 小格。計數時,4 大格均計數,總數除以 4(得每大格細胞數),再乘以 10(10 倍稀釋),即為實際 n 萬/mL 細胞濃度。傳代當天記為第一天,若第二天進行轉染,鋪 900-1000 萬/T75;若第三天轉染,鋪 350-400 萬/T75。每瓶 T75 加 10mL 10%DMEM 培養基。轉染當天觀察細胞密度,80-90%滿即可進行轉染。轉染前無需換培養基。
 
  • 做脂轉 complex
 
Opti MEM 需在 37 度水浴中預熱,Lipofiter
TM轉染試劑需恢復至室溫方可使
用,使用前需搖勻。
轉染每瓶 T75 的 complex 成分如下:
pspax  10μg
PMD2G  10μg
pTSB系列載體  10μg
轉染后 6h 換新鮮培液。
 
  • 病毒收集
 
轉染后 48 和 72h 分別兩次收集病毒上清(24h后置換新鮮培液),收集
后以 0.45 μm 濾器過濾,于 40 mL 超速離心管中,4℃,72000g/min 離心 120 分
鐘;
  • 病毒重懸和保存
 
500ul 新鮮培養液重懸病毒沉淀,置于-80 度甚至液氮保存。
 
五、感染目的細胞
 
  • 細胞準備
 
將狀態良好的目的細胞接種到 24 孔板,使細胞濃度為 1×105/ml 細胞,接種細胞數量因細胞的生長速度而略有不同,一般是保證第二天進行病毒感染的時候細胞匯合率介于 50%至 70%直接。
 
  • 病毒感染
 
  1. polybrene 的選擇:
 
Polybrene:是帶正電的小分子,與細胞表面的陰離子結合,提高慢病毒對細
胞的感染效率,通常加入 polybrene 能提高感染效率 2~10 倍。
Polybrene 有一定的細胞毒性,有的細胞對 polybrene 的毒理反應明顯,因此細胞感染時是否適合 polybrene 需要摸索;不同細胞對 polybrene 的敏感度不同,可以用 1~10μg/ml 的范圍篩選合適的濃度,以 24h 內細胞無明顯毒性反應為佳,可參考文獻并進行預實驗摸索,polybrene 最常用的工作濃度為 6~8μg/ml。
 
2. 感染細胞最佳 MOI 的測定
 
MOI(Multiplicity of Infection,感染復數)是指每個細胞感染的病毒
數,通常 MOI 越高,病毒整合到染色體的數量以及目的蛋白的表達量越高。
對于分裂活躍的細胞,比如 Hela、293 細胞,MOI=1~3 時,80%以上的細胞均表達目的基因。而對于非分裂細胞,比如原代細胞,感染效率較低。需要進行 MOI梯度摸索實驗,選擇適合的 MOI 進行實驗。
 
3.感染步驟
 
按實驗需要將細胞鋪板(比如 12 孔板)。細胞數以第 2 天密度約50%為宜。
37℃ 培養過夜。 對于 Polybrene 可施加的細胞:準備完全培養基和 Polybrene 混合物,Polybrene終濃度為摸索得到的最適終濃度。移去培養基并添加 0.5  ml Polybrene/培養基混合物于每孔中(適用于 24 孔板,其他孔板請相應調整體積。) 對于不適用 Polybrene 的細胞,以上步驟省略,直接進入下面步驟。感染前,從冰箱取出并在 37℃水浴中快速融化病毒,吸去細胞原有培養基,將病毒原液加入細胞中,輕輕 8 字混勻。(具體加入的病毒數可參考附表 1)感染后第二天(約 12 小時),吸去含病毒的培養液,換上新鮮的完全培養液,繼續 37℃培養。感染后 48 小時,對于帶 GFP 報告基因的病毒,可通過熒光顯微鏡觀測 GFP表達效率,對于攜帶 Puromycin 抗性基因的病毒,換上含適當濃度的 Puromycin的新鮮完全培養液,篩選穩定轉導的細胞株(詳見下方)。
注意: 溶化的 shRNA 慢病毒顆粒置于冰上。反復凍溶或長時間將病毒顆粒暴露于常溫可使病毒效價降低。
 
4.Puromycin 抗性篩選:
 
Puromycin 標準的施加終濃度范圍為 1~10μg/ml,不同細胞 puromycin 的工作濃度不同,請查找相關文獻(部分細胞參考用量見下圖),另外請設不感染篩選對照(未經過病毒感染的野生型細胞),加入等量等濃度的 puromycin。
 
 
部分細胞 Species puromycin參考值
Cell line Puromycin(µg/ml)
293 Human 3
HeLa Human 3
B16 Mouse 1至3
PC1.0 Hamster 10
MC3T3-E1 Mouse 10
H9C2 Rat 1
MCF-7 Human 1至3
MDA-MB-××× Human 1至3
HepG2 Human 2
HT1080 Human 1
A549 Human 1.5
H1299 Human 2
 
更多Puromycin使用濃度更新中,詳情請參考公司網站……
每 2-3 天換含 puromycin 的完全培養液一次,至不感染篩選對照組細胞被
puromycin 殺光,可進行以下兩步操作(依照具體實驗需要選擇)。
不挑取單克隆:
將感染并篩選后的細胞進行傳代,并繼續施加 puromycin 進行維持性篩選培養。連續篩選并傳 3 代后,凍存保重穩定細胞株。
挑取單克隆:
將感染并篩選后的細胞挑選至少 5 個克隆進行細胞擴增,,并繼續施加 puromycin進行維持性篩選培養。擴增完畢后 western blot 或者 QPCR 檢測目的蛋白的表達。挑取表達適中的穩定細胞株,連續篩選并傳 3 代后,凍存保重穩定細胞株。
 
5. 感染懸浮細胞
 
上面介紹的是針對貼壁細胞的感染方法,若是懸浮或半懸浮細胞,則需要通
過平角離心轉染法,即將適量的病毒液加入細胞培養皿后,封好口,放入平角離心機后,低速(500g-1000g/min)離心1h,然后放入培養箱中正常培養即可。若由于實驗條件有限,沒有平角離心機,可用離心管代替,將細胞吹打吸入離心管中,進行低速離心,去掉大部分上清,然后加入適量的病毒液,室溫放置15min(不能超過半小時),然后將細胞和病毒液同時吸出轉入培養皿中繼續病毒感染過夜后換液即可。
 
6. 對于極難感染的細胞
 
對于極難感染的細胞,如DC(樹突狀細胞)等,可采用多次感染的方法,
即感染24小時后,更換新鮮病毒進行二次感染,可顯著提升感染效率。
 
 
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