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  MicroRNA（miRNA）是一類內(nèi)生的、長(zhǎng)度約20-24個(gè)核苷酸的小RNA,是發(fā)夾結(jié)構(gòu)的約70-90個(gè)堿基大小的單鏈RNA前體經(jīng)過(guò)Dicer酶加工后生成。其在細(xì)胞內(nèi)具有多種重要的調(diào)節(jié)作用。每個(gè)miRNA可以有多個(gè)靶基因，而幾個(gè)miRNAs也可以調(diào)節(jié)同一個(gè)基因。這種復(fù)雜的調(diào)節(jié)網(wǎng)絡(luò)既可以通過(guò)一個(gè)miRNA來(lái)調(diào)控多個(gè)基因的表達(dá)，也可以通過(guò)幾個(gè)miRNAs的組合來(lái)精細(xì)調(diào)控某個(gè)基因的表達(dá)。據(jù)推測(cè)，miRNA調(diào)節(jié)著人類三分之一的基因。

    約70bp含microRNA莖環(huán)結(jié)構(gòu)的pre-miRNA。

    pri－miRNA

    天然pri－miRNA ：從染色體基因文庫(kù)中調(diào)取300bp－1000bp完整的microRNA基因，克隆到質(zhì)粒載體（普通載體或病毒載體），以強(qiáng)大的CMV啟動(dòng)子操 縱該300bp－1000bp microRNA。

    人工pri－miRNA： 選擇一個(gè)完整的microRNA基因，克隆到質(zhì)粒載體（普通載體或病毒載體）。以人工合成的約70bp含miRNA莖環(huán)結(jié)構(gòu)的目標(biāo)pre-RNA替代原pre-RNA，并以pol Ⅱ/pol III 啟動(dòng)子操縱該microRNA結(jié)構(gòu)單元。

 

  pre-miRNA是最早采用的microRNA，擁有大量的成功報(bào)道。化學(xué)合成的pre-miRNA制備快捷，可以標(biāo)記熒光等追蹤。缺點(diǎn)是制備的RNA穩(wěn)定性較差，而且，由于制備的microRNA較長(zhǎng)，合成時(shí)RNA的錯(cuò)誤難以避免（當(dāng)合成RNA超過(guò)60bp時(shí)，出現(xiàn)一個(gè)堿基錯(cuò)誤率約30％），轉(zhuǎn)錄制備的pre-miRNA穩(wěn)定性較好，但無(wú)flank結(jié)構(gòu)，很少使用。質(zhì)粒載體形式的pre－miRNA也因?yàn)榘l(fā)現(xiàn)microRNA的flank對(duì)于microRNA功能和測(cè)定非常重要，現(xiàn)已逐漸被pri-miRNA取代。人工pri-miRNA效果較pre-miRNA好，也有足夠多的成功使用的經(jīng)驗(yàn)報(bào)道，但這種人工microRNA采用固定的mir155 flank，制備的microRNA功能不及天然原始microRNA，故也逐漸較少使用。原始天然pri-miRNA克隆自天然文庫(kù)的microRNA由于保留了每個(gè)microRNA獨(dú)自的原始天然雙臂結(jié)構(gòu)，效果更好，是近來(lái)被首選方法。

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

我們采用美國(guó)進(jìn)口的第三代microRNA過(guò)表達(dá)慢病毒表達(dá)載體，有HIV來(lái)源的和FIV來(lái)源兩種，能夠高效表達(dá)出原始天然pri-miRNA和MicroRNA基因簇，詳情請(qǐng)參見(jiàn)慢病毒載體信息，我們的microRNA過(guò)表達(dá)慢病毒載體具有以下特點(diǎn)：

 

 

 

 

 

 

 

 

 

 

       3 ΔLTR (ΔU3)

       3’ LTR區(qū)域刪除了U3，使其不再有啟動(dòng)/增強(qiáng)活性，成為自滅活（self-inactivating, SIN） 載體，即整合到細(xì)胞基因組后，不會(huì)產(chǎn)生新的子代病毒，也降低了對(duì)周圍基因的意外激活。

       copGFP

綠色熒光報(bào)告基因，與常用的EGFP類似，但熒光更亮。

       Zeo抗性基因

陽(yáng)性細(xì)胞篩選。

       pUC起始點(diǎn)

使質(zhì)粒在 E.coli 中高密度拷貝并且保持穩(wěn)定。

       RSV/5’LTR （RSV雜交啟動(dòng)子-R/U5’長(zhǎng)末端重復(fù)序列

讓全長(zhǎng)病毒在293前體細(xì)胞中高水平包裝和轉(zhuǎn)錄表達(dá)。

       CMV/5’LTR （CMV雜交型啟動(dòng)子-R/U5長(zhǎng)末端重復(fù)序列）

使慢病毒載體保持高水平轉(zhuǎn)錄和高效轉(zhuǎn)錄出含病毒包裝必需功能元件（如Psi, RRE, and cPPT等)的轉(zhuǎn)錄本，當(dāng)用病毒包裝細(xì)胞系（如HEK 293細(xì)胞）包裝病毒時(shí)也能表達(dá)病毒衣殼蛋白。

       SV40起始點(diǎn)

使質(zhì)粒在哺乳動(dòng)物細(xì)胞中穩(wěn)定復(fù)制。

       SV40多聚A加尾信號(hào)

強(qiáng)制轉(zhuǎn)錄終止子，有效終止轉(zhuǎn)錄或者終止共轉(zhuǎn)錄過(guò)程，維持穩(wěn)定態(tài)mRNA高水平表達(dá)。

       LCS無(wú)連接酶克隆位點(diǎn)

不依賴多克隆位點(diǎn)，能將各種目的microRNA插入載體，不需要DNA鏈接酶，較常規(guī)方法可靠。

       WPRE元件

增強(qiáng)CMV啟動(dòng)轉(zhuǎn)錄的穩(wěn)定性。

 

說(shuō)明：每種載體用的優(yōu)勢(shì)元件均有所不同 

  

             

  目前我們能夠?yàn)榭蛻籼峁┌�括miRNA表達(dá)譜分析、慢病毒載體構(gòu)建、測(cè)序、慢病毒生產(chǎn)等一站式服務(wù)。我們的服務(wù)將為客戶最大限度地提高效率和節(jié)省時(shí)間。另外，我們的技術(shù)專家將為您的實(shí)驗(yàn)提供全程支持，請(qǐng)Email至[email protected]。

  同時(shí)，我們?yōu)榭蛻籼峁﹎iRNA過(guò)表達(dá)慢病毒載體構(gòu)建服務(wù)的個(gè)性化定制服務(wù)方案，包括根據(jù)miRNA特征、目的細(xì)胞特征與客戶需求，我們會(huì)為客戶選擇最合適的慢病毒載體、啟動(dòng)子、熒光標(biāo)記或選擇性抗性標(biāo)記，插入pri-miRNA或miRNA基因簇使其過(guò)表達(dá)，客戶也可以根據(jù)我們的載體信息自行挑選；同時(shí)又可以根據(jù)客戶的需要，可在重組慢病毒載體中加入各種報(bào)告基因或標(biāo)簽（或進(jìn)行改造），從而構(gòu)建miRNA過(guò)表達(dá)慢病毒載體。

  我們有標(biāo)準(zhǔn)的客制化服務(wù)流程，同時(shí)我們也會(huì)根據(jù)客戶需求，為每個(gè)客戶度身定制各種MicroRNA慢病毒載體構(gòu)建服務(wù)方案。如有需要，歡迎致電021-34512321咨詢。

 

 

 

 

  我們可以根據(jù)客戶需求構(gòu)建各種濃度的超純純化miRNA過(guò)表達(dá)慢病毒載體，能被包裝成miRNA過(guò)表達(dá)慢病毒顆粒；無(wú)內(nèi)源性多聚A信號(hào)；對(duì)靶細(xì)胞無(wú)毒；從5 to 3 LTR序列大小大于8kb；可直接轉(zhuǎn)染細(xì)胞，用于瞬時(shí)轉(zhuǎn)染；也可以包裝成病毒顆粒，用于穩(wěn)定長(zhǎng)效表達(dá)。

 

                   內(nèi)毒素<10EU/mg質(zhì)粒DNA

                   RNA<0.2ug/mg

                   基因組DNA<2ug/mg

                   蛋白<3ug/mg 

 

 

 

 

 

 

 

 

 

  用HIV來(lái)源的慢病毒載體構(gòu)建的MicroRNA Lenti-miR載體包裝成病毒顆粒之后，感染HEK293細(xì)胞，表達(dá)的MicroRNA用qPCR檢測(cè)，結(jié)果發(fā)現(xiàn)用我們的過(guò)表達(dá)載體表達(dá)出MicroRNA是內(nèi)源性MicroRNA的幾倍以上。

 

  

  microRNA(miRNA)基因簇是一類miRNA基因在染色體上成簇排列形成的基因群. 在動(dòng)植物基因組中，已發(fā)現(xiàn)存在大量簇生排列的miRNA基因。然而這種簇生排列方式所具有的功能意義尚不完全清楚. miRNA基因簇常位于一個(gè)多順?lè)醋觾?nèi), 通過(guò)共表達(dá)在胚胎發(fā)育、細(xì)胞周期、腫瘤發(fā)生、細(xì)胞分化等諸多方面發(fā)揮協(xié)同調(diào)控的功能。

  詳細(xì)的構(gòu)建服務(wù)流程及其他信息請(qǐng)見(jiàn)上述microRNA慢病毒載體構(gòu)建服務(wù)內(nèi)容。

 

 

 

 

 

 

miR-10b construct cloned using Clone-it microRNA Cloning System was transiently transfected into 293 cells with the indicated amount of plasmid DNA.  Mature miR-10b was measured using qPCR

  

 

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