哺乳动物miR30-shRNA干扰慢病毒载体

慢病毒miR30 shRNA干扰载体系统是用于多种哺乳动物细胞中，高效干扰靶基因表达的方法。当病毒基因组被逆转录并永久整合到宿主细胞基因组时，用户定制的启动子会驱动包含目的基因和一个或多个基于miR30的靶向目的基因的shRNA（shRNAmiR）多顺反子的表达。 shRNAmiR转录物通过胞内micro-RNA途径加工产生成熟的shRNA，促进靶基因mRNA的降解。

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慢病毒miR30 shRNA干扰载体使用优化的micro-RNA系统，衍生自第三代慢病毒载体。经优化，该载体在大肠杆菌体内具有很高的拷贝数，包装的活病毒具有很高的滴度，对大多数宿主细胞具有高效的转导能力，能有效地把载体整合到靶细胞基因组并实现外源基因的高水平表达。用户定制的启动子可以驱动包含目的基因和一个或多个基于miR30的靶向目的基因的shRNA（shRNAmiR）的表达，从而介导有效的shRNA加工和靶基因干扰。

图1 miR30 shRNA慢病毒载体系统的EGFP敲低和mCherry表达效果。（A）将含有CMV启动子驱动的mCherry和打靶EGFP的miR30 shRNA的慢病毒载体包装成慢病毒颗粒。对照慢病毒载体具有相同的载体结构，但是使用了scramble shRNA。用这些病毒转导表达EGFP的HEK293T细胞。药筛细胞后，使用流式细胞术和荧光显微镜测定EGFP和mCherry的表达。(B) 荧光图像和中值荧光强度 (图中的 MFI ± SD) 表明，与scramble相比，靶向 EGFP 的 shRNA 显着降低了 EGFP 表达 (P<0.001)。此外，mCherry在任一一种经过慢病毒转导的细胞中均表达，但在亲本HEK293T-EGFP细胞中不表达。这些结果表明，目的基因和miR30 shRNA 都可以在pol II 启动子的驱动下共表达。每组实验重复三次，数据经过双尾t检验分析。

启动子选择多样：与利用RNA聚合酶III启动子如U6的标准shRNA系统不同，基于miR30的shRNA可以通过多种RNA聚合酶II启动子转录，可以使用组织特异性或诱导型启动子。

多个shRNA共表达：由于RNA聚合酶II能够有效转录长RNA，因此多个shRNAmiR可以作为多顺反子由单个启动子驱动表达。

可与报告基因ORF共表达：用户选择的目的基因或报告基因ORF与shRNAmiRs作为多顺反子一起共表达，有助于监测shRNA的转录。

永久性干扰：慢病毒整合到宿主细胞基因组是一个不可逆的过程，对于靶基因的干扰通常是稳定和永久的。 基于这个优势，可对培养细胞或活体干扰表型进行长期分析，有助于分离具有不同干扰水平和/或不同表型的克隆；当干扰载体携带荧光标记如EGFP时，可通过流式分选具有不同荧光强度（荧光强度和整合数量有关，进而与干扰程度有关）的细胞。

滴度高：我们的病毒载体可以包装出高滴度的病毒。我们提供的病毒包装服务，病毒滴度可以达到>10⁹ TU/ml。在这样的病毒滴度下，如果选择合适的剂量去转导体外培养的哺乳动物细胞，则转导效率可接近100%。

宿主范围广泛：我们的病毒包装系统包装出来的病毒含有VSV-G包膜蛋白，此蛋白拥有非常广泛的亲和性，可以转导几乎所有的哺乳动物细胞，包括分裂细胞，非分裂细胞，原代细胞，稳定细胞系，干细胞，分化细胞，贴壁细胞和悬浮细胞等各类哺乳动物细胞，甚至还可以转导一些非哺乳动物细胞。使用传统的转染方式转导神经元细胞是非常难的，但是采用我们慢病毒载体系统可以轻易的实现神经元细胞的转导。相对于在某些细胞中具有较低转导效率的腺病毒和不能用于非分裂细胞的逆转录病毒而言，利用我们的慢病毒包装系统包装出来的病毒具有广泛的亲和性。

基因拷贝数相对均一：通常情况下，采用病毒转导的方式可以比较均一的将外源基因转入靶细胞中，而传统的质粒转染则呈现出较高的不均一性，导致某些细胞会获得较多拷贝质粒而某些则会获得较少甚至完全没有。

体内外实验均有效：我们的载体不仅拥有良好的体外细胞转导能力，同样适用于体内活体动物实验。

安全性：我们的病毒载体系统具备了以下两大特点，因而具有非常高的安全性。一、病毒包装和转导所必需的基因由三个辅助质粒分开表达。二、5' LTR的启动子自失活。因此，在进行病毒包装和病毒转导的时候不会产生具有复制能力的病毒颗粒，使用我们的载体对人体的健康威胁也是最低的。

启动子干扰效应：启动子强度、shRNA表达水平以及干扰效果之间存在很大的相关性。许多RNA聚合酶II启动子比U6 RNA聚合酶III强启动子表达shRNA的能力更弱，这会导致靶基因干扰效果下降。

技术复杂：使用慢病毒载体时，需要在包装细胞中产生活病毒，然后测定病毒滴度。因此慢病毒转染相对于常规质粒转染，技术难度更高，周期更长

永久性干扰：慢病毒整合到宿主细胞基因组是一个不可逆的过程，如果使用的是组成型启动子，对于靶基因的干扰通常是稳定和永久的。 一旦慢病毒shRNA干扰载体对基因产生了干扰，目的基因的表达就很难被重新激活。 根据不同的实验目的，这有可能是优势也可能是劣势。

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.