IPTG诱导shRNA干扰慢病毒载体

IPTG诱导shRNA干扰慢病毒载体是一种非常高效的、可在多种哺乳动物细胞中实现临时性目的基因敲低的系统，是研究细胞发育、细胞生存必需基因的一套有效工具。该系统利用来自细菌乳糖操纵子的LacI（抑制因子）和LacO（操作因子）蛋白之间的相互作用，在乳糖类似物IPTG（异丙基-ß-D-硫代半乳糖苷）存在或者缺乏的情况下调控shRNA的表达。

我们的IPTG诱导shRNA干扰慢病毒载体采用的是第三代慢病毒载体系统。该系统经过改造优化，在大肠杆菌体内具有较高的拷贝数，包装的活病毒具有较高的滴度，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。在IPTG存在下，可阻止LacI与LacO相结合，经过修饰的携带两个LacO元件的U6启动子在哺乳动物细胞中高效驱动下游shRNA高水平的表达。我们经过优化的shRNA茎-环序列可介导高效的shRNA加工和靶基因敲低。

图1所示为我们的IPTG诱导干扰慢病毒载体在IPTG存在条件下高效的靶基因敲低效率。

图1： IPTG诱导慢病毒载体系统对EGFP的敲低效率。 (A) 基于U6的IPTG诱导慢病毒shRNA scramble载体或者靶向EGFP的shRNA载体分别包装到慢病毒后，并转导到EGFP稳定表达的HEK293T细胞株中。然后选择适当的抗生素，如puromycin (Puro) 或者blasticidin (Bsd)等，筛选阳性转导细胞，随后用1mM IPTG诱导shRNA表达。最后使用流式细胞法（FCM）对所有实验组的EGFP的中位荧光强度（MFI）进行定量。 (B) 经IPTG诱导后，表达诱导EGFP shRNA的细胞EGFP MFI降低42%。该结果在携带Puro和Bsd抗性基因的诱导shRNA载体中表现一致。表达非打靶scramble shRNA的诱导shRNA载体，IPTG的诱导对EGFP MFI没有影响。此外，缺乏LacI抑制子的诱导shRNA载体转导的细胞，载体的诱导功能丧失，在有或者没有IPTG的条件下，EGFP的表达均受到EGFP shRNA的组成性抑制。

严格调控和高诱导： U6启动子携带的两个重复的LacO元件和shRNA下游额外的LacO元件，在没有IPTG的条件下，可以让背景启动子的活性最小化；在IPTG存在的条件下，可使基因沉默最大化，从而实现了对shRNA表达的严格调控。

响应快速： IPTG诱导基因沉默可快至48h。

更高效率：: 该系统比Tet依赖的诱导敲低系统可以实现更高效和更加动态的敲低效果。 

高病毒滴度： 我们的病毒载体可以出高滴度的病毒，我们提供的病毒包装服务，病毒滴度可达到>10^9 TU/ml。在这样的病毒滴度下，如果使用合适的病毒剂量去转导培养哺乳动物细胞，转导效率可接近100%。

广泛的亲嗜性： 我们的病毒包装系统在病毒表面添加VSV-G衣壳蛋白，该蛋白有广泛的亲嗜性，因此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困难的神经元细胞，采用我们的慢病毒载体系统可以轻易实现转导。相对于在某些细胞中转导效率较低的腺病毒或者不能用于非分裂细胞的MMLV逆转录病毒而言，我们的慢病毒包装系统包装出来的病毒具有广泛的亲嗜性。

载体拷贝数相对均一： 通常情况下，采用病毒转导的方式可以相对均一地将外源基因转入到靶细胞中，而传统的质粒转染方式则呈现出高度不均一性，导致某些细胞会获得较多的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： 我们的慢病毒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。其一，病毒包装和转导所必需的基因由三个辅助质粒分开表达。其二，5' 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风险也是最小的。

技术复杂性: 使用慢病毒载体需要在包装细胞中生产病毒并进行病毒滴度检测，因此比传统的质粒转染方法，技术难度更高，周期更长。

RSV promoter: Rous sarcoma virus promoter. It drives transcription of viral RNA in packaging cells. This RNA is then packaged into live virus.

5' LTR-ΔU3: 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.

U6/2xLacO: Modified human U6 small nuclear 1 promoter containing two repeats of the lac operator sequence. Pol III promoter which suppresses small RNA expression in the presence of LacI.

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

Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.

Terminator: Terminates transcription of the shRNA.

LacO: Lac operator. In the absence of IPTG, it is bound by lac repressor (LacI), leading to transcriptional repression of the upstream shRNA. In the presence of IPTG, LacI can no longer bind to LacO, thus allowing upstream shRNA to be transcribed.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream ORF.

Regulatory protein: Lac repressor (LacI) and a drug selection gene (such as puromycin resistance gene) linked by T2A linker. Allows cells to express LacI protein and be resistant to the corresponding drug. In the absence of IPTG, LacI binds to LacO to repress transcription of downstream genes or small RNAs. In the presence of IPTG, LacI undergoes a conformational change and is no longer able to bind to LacO, thus allowing downstream genes or small RNAs to be transcribed.

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

3' LTR-ΔU3: 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.