慢病毒miR30 shRNA干擾載體系統(tǒng)是用于多種哺乳動物細胞中，高效干擾靶基因表達的方法。當病毒基因組被逆轉(zhuǎn)錄并永久整合到宿主細胞基因組時，用戶定制的啟動子會驅(qū)動包含目的基因和一個或多個基于miR30的靶向目的基因的shRNA（shRNAmiR）多順反子的表達。 shRNAmiR轉(zhuǎn)錄物通過胞內(nèi)micro-RNA途徑加工產(chǎn)生成熟的shRNA，促進靶基因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，對大多數(shù)宿主細胞具有高效的轉(zhuǎn)導能力，能有效地把載體整合到靶細胞基因組并實現(xiàn)外源基因的高水平表達。用戶定制的啟動子可以驅(qū)動包含目的基因和一個或多個基于miR30的靶向目的基因的shRNA（shRNAmiR）的表達，從而介導有效的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表達效果。（A）將含有CMV啟動子驅(qū)動的mCherry和打靶EGFP的miR30 shRNA的慢病毒載體包裝成慢病毒顆粒。對照慢病毒載體具有相同的載體結構，但是使用了scramble shRNA。用這些病毒轉(zhuǎn)導表達EGFP的HEK293T細胞。藥篩細胞后，使用流式細胞術和熒光顯微鏡測定EGFP和mCherry的表達。(B) 熒光圖像和中值熒光強度 (圖中的 MFI ± SD) 表明，與scramble相比，靶向 EGFP 的 shRNA 顯著降低了 EGFP 表達 (P<0.001)。此外，mCherry在任一一種經(jīng)過慢病毒轉(zhuǎn)導的細胞中均表達，但在親本HEK293T-EGFP細胞中不表達。這些結果表明，目的基因和miR30 shRNA 都可以在pol II 啟動子的驅(qū)動下共表達。每組實驗重復三次，數(shù)據(jù)經(jīng)過雙尾t檢驗分析。

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

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

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

永久性干擾：慢病毒整合到宿主細胞基因組是一個不可逆的過程，對于靶基因的干擾通常是穩(wěn)定和永久的。 基于這個優(yōu)勢，可對培養(yǎng)細胞或活體干擾表型進行長期分析，有助于分離具有不同干擾水平和/或不同表型的克?。划敻蓴_載體攜帶熒光標記如EGFP時，可通過流式分選具有不同熒光強度（熒光強度和整合數(shù)量有關，進而與干擾程度有關）的細胞。

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，病毒滴度可以達到>10⁹ TU/ml。在這樣的病毒滴度下，如果選擇合適的劑量去轉(zhuǎn)導體外培養(yǎng)的哺乳動物細胞，則轉(zhuǎn)導效率可接近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)導幾乎所有的哺乳動物細胞，包括分裂細胞，非分裂細胞，原代細胞，穩(wěn)定細胞系，干細胞，分化細胞，貼壁細胞和懸浮細胞等各類哺乳動物細胞，甚至還可以轉(zhuǎn)導一些非哺乳動物細胞。使用傳統(tǒng)的轉(zhuǎn)染方式轉(zhuǎn)導神經(jīng)元細胞是非常難的，但是采用我們慢病毒載體系統(tǒng)可以輕易的實現(xiàn)神經(jīng)元細胞的轉(zhuǎn)導。相對于在某些細胞中具有較低轉(zhuǎn)導效率的腺病毒和不能用于非分裂細胞的逆轉(zhuǎn)錄病毒而言，利用我們的慢病毒包裝系統(tǒng)包裝出來的病毒具有廣泛的親和性。

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

體內(nèi)外實驗均有效：我們的載體不僅擁有良好的體外細胞轉(zhuǎn)導能力，同樣適用于體內(nèi)活體動物實驗。

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

啟動子干擾效應：啟動子強度、shRNA表達水平以及干擾效果之間存在很大的相關性。許多RNA聚合酶II啟動子比U6 RNA聚合酶III強啟動子表達shRNA的能力更弱，這會導致靶基因干擾效果下降。

技術復雜：使用慢病毒載體時，需要在包裝細胞中產(chǎn)生活病毒，然后測定病毒滴度。因此慢病毒轉(zhuǎn)染相對于常規(guī)質(zhì)粒轉(zhuǎn)染，技術難度更高，周期更長

永久性干擾：慢病毒整合到宿主細胞基因組是一個不可逆的過程，如果使用的是組成型啟動子，對于靶基因的干擾通常是穩(wěn)定和永久的。 一旦慢病毒shRNA干擾載體對基因產(chǎn)生了干擾，目的基因的表達就很難被重新激活。 根據(jù)不同的實驗目的，這有可能是優(yōu)勢也可能是劣勢。

RSV promoter: Rous sarcoma virus 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 RSV 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.

mPGK promoter: Mouse phosphoglycerate kinase 1 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.