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                                                                                                              shRNA載體構建

服務介紹

shRNA(short hairpin RNA, shRNA)是一段小發卡或短發卡RNA序列,一般長19~23bp,常被用于RNA干擾沉默靶基因的表達。利用載體把shRNA導入細胞,載體中的U6或H1啟動子確保shRNA的表達;shRNA的發卡結構可被細胞機制切割成siRNA,然后siRNA結合到RNA誘導沉默復合物上(RNA induce
-d silencing complex,RISC),該復合物能夠結合到目的mRNAs并將其降解。通過載體可以將shRNA整合到細胞基因組中,從而使細胞長期穩定表達shRNA。
權陽生物可提供多種shRNA表達載體,如慢病毒載體、腺病毒載體、腺相關病毒載體、以及普通的真核表達載體等;同時也有多種熒光報告基因可供選擇,如GFP、eGFP、tdTomato、YEP等;另外還有藥篩基因可供選擇:Puromycin、Neomycin等,用于后期構建穩定表達shRNA的細胞株。

服務流程

1、接受客戶目標基因信息; 
2、分析目標基因,并設計高質量的shRNA序列(客戶可從Transheep shRNA 文庫中自行選擇序列); 
3、PCR退火形成shRNA雙鏈; 
4、將shRNA雙鏈克隆至含有U6或H1啟動子的表達載體上; 
5、測序以確保插入序列的正確性; 
6、向客戶提交測序結果與實驗方法報告單。

載體選擇(可到Transheep 質???/span>中選擇相應載體)

載體舉例
<
慢病毒載體   腺病毒載體
plko.1-TRC   pShuttle-U6
plko.1 puro   pShuttle-U6-CMV-RFP
plko.3G   pShuttle-U6-CMV-EGFP
pLVX-sh1    
pLKO-puro-TurboGFP    
pLVX-sh2    
pLVX-U6-RFP-Puro    
pLVX-U6-GFP-Puro    
pLVX-U6-GFP    
pLVX-U6-tdTomato    
pLVX-U6-YFP    
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