慢病毒miR30 shRNA干擾載體系統(tǒng)是用于多種哺乳動(dòng)物細(xì)胞中，高效干擾靶基因表達(dá)的方法。當(dāng)病毒基因組被逆轉(zhuǎn)錄并永久整合到宿主細(xì)胞基因組時(shí)，用戶定制的啟動(dòng)子會(huì)驅(qū)動(dòng)包含目的基因和一個(gè)或多個(gè)基于miR30的靶向目的基因的shRNA（shRNAmiR）多順反子的表達(dá)。 shRNAmiR轉(zhuǎn)錄物通過(guò)胞內(nèi)micro-RNA途徑加工產(chǎn)生成熟的shRNA，促進(jìn)靶基因mRNA的降解。

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慢病毒miR30 shRNA干擾載體使用優(yōu)化的micro-RNA系統(tǒng)，衍生自第三代慢病毒載體。經(jīng)優(yōu)化，該載體在大腸桿菌體內(nèi)具有很高的拷貝數(shù)，包裝的活病毒具有很高的滴度，對(duì)大多數(shù)宿主細(xì)胞具有高效的轉(zhuǎn)導(dǎo)能力，能有效地把載體整合到靶細(xì)胞基因組并實(shí)現(xiàn)外源基因的高水平表達(dá)。用戶定制的啟動(dòng)子可以驅(qū)動(dòng)包含目的基因和一個(gè)或多個(gè)基于miR30的靶向目的基因的shRNA（shRNAmiR）的表達(dá)，從而介導(dǎo)有效的shRNA加工和靶基因干擾。

圖1 miR30 shRNA慢病毒載體系統(tǒng)的EGFP敲低和mCherry表達(dá)效果。（A）將含有CMV啟動(dòng)子驅(qū)動(dòng)的mCherry和打靶EGFP的miR30 shRNA的慢病毒載體包裝成慢病毒顆粒。對(duì)照慢病毒載體具有相同的載體結(jié)構(gòu)，但是使用了scramble shRNA。用這些病毒轉(zhuǎn)導(dǎo)表達(dá)EGFP的HEK293T細(xì)胞。藥篩細(xì)胞后，使用流式細(xì)胞術(shù)和熒光顯微鏡測(cè)定EGFP和mCherry的表達(dá)。(B) 熒光圖像和中值熒光強(qiáng)度 (圖中的 MFI ± SD) 表明，與scramble相比，靶向 EGFP 的 shRNA 顯著降低了 EGFP 表達(dá) (P<0.001)。此外，mCherry在任一一種經(jīng)過(guò)慢病毒轉(zhuǎn)導(dǎo)的細(xì)胞中均表達(dá)，但在親本HEK293T-EGFP細(xì)胞中不表達(dá)。這些結(jié)果表明，目的基因和miR30 shRNA 都可以在pol II 啟動(dòng)子的驅(qū)動(dòng)下共表達(dá)。每組實(shí)驗(yàn)重復(fù)三次，數(shù)據(jù)經(jīng)過(guò)雙尾t檢驗(yàn)分析。

啟動(dòng)子選擇多樣：與利用RNA聚合酶III啟動(dòng)子如U6的標(biāo)準(zhǔn)shRNA系統(tǒng)不同，基于miR30的shRNA可以通過(guò)多種RNA聚合酶II啟動(dòng)子轉(zhuǎn)錄，可以使用組織特異性或誘導(dǎo)型啟動(dòng)子。

多個(gè)shRNA共表達(dá)：由于RNA聚合酶II能夠有效轉(zhuǎn)錄長(zhǎng)RNA，因此多個(gè)shRNAmiR可以作為多順反子由單個(gè)啟動(dòng)子驅(qū)動(dòng)表達(dá)。

可與報(bào)告基因ORF共表達(dá)：用戶選擇的目的基因或報(bào)告基因ORF與shRNAmiRs作為多順反子一起共表達(dá)，有助于監(jiān)測(cè)shRNA的轉(zhuǎn)錄。

永久性干擾：慢病毒整合到宿主細(xì)胞基因組是一個(gè)不可逆的過(guò)程，對(duì)于靶基因的干擾通常是穩(wěn)定和永久的。 基于這個(gè)優(yōu)勢(shì)，可對(duì)培養(yǎng)細(xì)胞或活體干擾表型進(jìn)行長(zhǎng)期分析，有助于分離具有不同干擾水平和/或不同表型的克隆；當(dāng)干擾載體攜帶熒光標(biāo)記如EGFP時(shí)，可通過(guò)流式分選具有不同熒光強(qiáng)度（熒光強(qiáng)度和整合數(shù)量有關(guān)，進(jìn)而與干擾程度有關(guān)）的細(xì)胞。

滴度高：我們的病毒載體可以包裝出高滴度的病毒。我們提供的病毒包裝服務(wù)，病毒滴度可以達(dá)到>10⁹ TU/ml。在這樣的病毒滴度下，如果選擇合適的劑量去轉(zhuǎn)導(dǎo)體外培養(yǎng)的哺乳動(dòng)物細(xì)胞，則轉(zhuǎn)導(dǎo)效率可接近100%。

宿主范圍廣泛：我們的病毒包裝系統(tǒng)包裝出來(lái)的病毒含有VSV-G包膜蛋白，此蛋白擁有非常廣泛的親和性，可以轉(zhuǎn)導(dǎo)幾乎所有的哺乳動(dòng)物細(xì)胞，包括分裂細(xì)胞，非分裂細(xì)胞，原代細(xì)胞，穩(wěn)定細(xì)胞系，干細(xì)胞，分化細(xì)胞，貼壁細(xì)胞和懸浮細(xì)胞等各類哺乳動(dòng)物細(xì)胞，甚至還可以轉(zhuǎn)導(dǎo)一些非哺乳動(dòng)物細(xì)胞。使用傳統(tǒng)的轉(zhuǎn)染方式轉(zhuǎn)導(dǎo)神經(jīng)元細(xì)胞是非常難的，但是采用我們慢病毒載體系統(tǒng)可以輕易的實(shí)現(xiàn)神經(jīng)元細(xì)胞的轉(zhuǎn)導(dǎo)。相對(duì)于在某些細(xì)胞中具有較低轉(zhuǎn)導(dǎo)效率的腺病毒和不能用于非分裂細(xì)胞的逆轉(zhuǎn)錄病毒而言，利用我們的慢病毒包裝系統(tǒng)包裝出來(lái)的病毒具有廣泛的親和性。

基因拷貝數(shù)相對(duì)均一：通常情況下，采用病毒轉(zhuǎn)導(dǎo)的方式可以比較均一的將外源基因轉(zhuǎn)入靶細(xì)胞中，而傳統(tǒng)的質(zhì)粒轉(zhuǎn)染則呈現(xiàn)出較高的不均一性，導(dǎo)致某些細(xì)胞會(huì)獲得較多拷貝質(zhì)粒而某些則會(huì)獲得較少甚至完全沒有。

體內(nèi)外實(shí)驗(yàn)均有效：我們的載體不僅擁有良好的體外細(xì)胞轉(zhuǎn)導(dǎo)能力，同樣適用于體內(nèi)活體動(dòng)物實(shí)驗(yàn)。

安全性：我們的病毒載體系統(tǒng)具備了以下兩大特點(diǎn)，因而具有非常高的安全性。一、病毒包裝和轉(zhuǎn)導(dǎo)所必需的基因由三個(gè)輔助質(zhì)粒分開表達(dá)。二、5' LTR的啟動(dòng)子自失活。因此，在進(jìn)行病毒包裝和病毒轉(zhuǎn)導(dǎo)的時(shí)候不會(huì)產(chǎn)生具有復(fù)制能力的病毒顆粒，使用我們的載體對(duì)人體的健康威脅也是最低的。

啟動(dòng)子干擾效應(yīng)：啟動(dòng)子強(qiáng)度、shRNA表達(dá)水平以及干擾效果之間存在很大的相關(guān)性。許多RNA聚合酶II啟動(dòng)子比U6 RNA聚合酶III強(qiáng)啟動(dòng)子表達(dá)shRNA的能力更弱，這會(huì)導(dǎo)致靶基因干擾效果下降。

技術(shù)復(fù)雜：使用慢病毒載體時(shí)，需要在包裝細(xì)胞中產(chǎn)生活病毒，然后測(cè)定病毒滴度。因此慢病毒轉(zhuǎn)染相對(duì)于常規(guī)質(zhì)粒轉(zhuǎn)染，技術(shù)難度更高，周期更長(zhǎng)

永久性干擾：慢病毒整合到宿主細(xì)胞基因組是一個(gè)不可逆的過(guò)程，如果使用的是組成型啟動(dòng)子，對(duì)于靶基因的干擾通常是穩(wěn)定和永久的。 一旦慢病毒shRNA干擾載體對(duì)基因產(chǎn)生了干擾，目的基因的表達(dá)就很難被重新激活。 根據(jù)不同的實(shí)驗(yàn)?zāi)康?，這有可能是優(yōu)勢(shì)也可能是劣勢(shì)。

CMV promoter: Human cytomegalovirus immediate early promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

Δ5' LTR: A deleted version of the HIV-1 5' long terminal repeat. In wildtype lentivirus, 5' LTR and 3' LTR are essentially identical in sequence. They reside on two ends of the viral genome and point in the same direction. Upon viral integration, the 3' LTR sequence is copied onto the 5' LTR. The LTRs carry both promoter and polyadenylation function, such that in wildtype virus, the 5' LTR acts as a promoter to drive the transcription of the viral genome, while the 3' LTR acts as a polyadenylation signal to terminate the upstream transcript. On our vector, Δ5' LTR is deleted for a region that is required for the LTR's promoter activity normally facilitated by the viral transcription factor Tat. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the CMV promoter engineered upstream of Δ5' LTR.

Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

RRE: HIV-1 Rev response element. It allows the nuclear export of viral RNA by the viral Rev protein during viral packaging.

cPPT: HIV-1 Central polypurine tract. It creates a "DNA flap" that increases nuclear importation of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

Promoter: Drives transcription of the downstream ORF and shRNAmiR polycistron. This is an RNA polymerase II promoter, rather than an RNA polymerase III promoter such as U6.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest or reporter gene is placed here. This can be used to monitor shRNA expression.

5' miR-30E: An optimized version of the human miR30 5’ context sequence. Facilitates maturation and processing of the shRNA and separation from the tandemly transcribed ORF and other shRNAs.

3' miR-30E: An optimized version of the human miR30 3’ context sequence. Facilitates maturation and processing of the shRNA and separation from the tandemly transcribed ORF and other shRNAs.

shRNAs: These sequences are derived from your target sequences, and are transcribed to form the stem portion of the “hairpin” structure of the shRNAs.

WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances viral RNA stability in packaging cells, leading to higher titer of packaged virus.

CMV promoter: Human cytomegalovirus immediate early promoter. This drives the ubiquitous expression of the downstream marker gene.

Marker: A drug selection gene (such as neomycin resistance), a visually detectable gene (such as EGFP), or a dual-reporter gene (such as EGFP/Neo). This allows cells transduced with the vector to be selected and/or visualized.

ΔU3/3' LTR: A truncated version of the HIV-1 3' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (due to the fact that 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in ΔU3/3' LTR serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

SV40 early pA: Simian virus 40 early polyadenylation signal. It further facilitates transcriptional termination after the 3' LTR during viral RNA transcription during packaging. This elevates the level of functional viral RNA in packaging cells, thus improving viral titer.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.