手机验证
云舟生物科技(广州)股份有限公司
提供定制载体
VectorBuilder的慢病毒诱导基因表达载体将高效的第三代慢病毒载体系统与Tet-On诱导基因表达系统相结合,帮助您实现四环素诱导基因表达盒与宿主基因组的永久整合。
Tet-On诱导基因表达系统是控制哺乳动物细胞中目的基因(GOI)表达时间的有力工具。我们的Tet-On诱导基因表达载体设计用于在没有四环素及其类似物(例如多西环素)的情况下实现GOI的几乎完全沉默,以及响应添加四环素或其类似物之一(例如强力霉素)的强烈快速表达。这是通过多组分系统实现的,该系统在没有四环素的情况下结合了tTS蛋白的活性沉默,以及在四环素存在下rtTA蛋白的强激活。在没有四环素的情况下,来自TetR(Tet阻遏蛋白)和KRAB-AB(Kid-1蛋白的转录抑制结构域)融合的tTS蛋白与TRE启动子结合,导致基因转录的主动抑制。另一方面,rtTA蛋白来源于突变的Tet阻遏因子和VP16(单纯疱疹病毒病毒蛋白16的转录激活结构域)的融合,仅在四环素存在下与TRE启动子结合以激活基因转录。
慢病毒载体系统是将基因永久引入哺乳动物细胞的高效载体。慢病毒载体首先在大肠杆菌中构建为质粒。对于慢病毒诱导基因表达载体,在载体构建过程中,由驱动GOI的四环素诱导元件(TRE)启动子组成的四环素诱导表达盒放置在两个LTR之间。然后将其与几个辅助质粒一起转染到包装细胞中。在包装细胞内,位于两个长末端重复序列(LTR)之间的载体DNA被转录成RNA,辅助质粒表达的病毒蛋白进一步将RNA包装成病毒。然后将活病毒释放到上清液中,上清液可用于直接感染靶细胞或在浓缩后感染。
当病毒被添加到靶细胞中时,RNA货物被穿梭到细胞中,在那里它被逆转录成DNA,并随机整合到宿主基因组中。在载体构建过程中放置在两个LTR之间的诱导表达盒与病毒基因组的其余部分一起永久插入宿主DNA。
虽然我们的慢病毒诱导基因表达载体包括由驱动用户选择的GOI的TRE启动子组成的诱导基因表达盒,但TRE结合调节蛋白rTS和rtTA必须使用单独的辅助载体提供,以便在四环素存在下实现四环素诱导的基因表达,同时在没有四环素的情况下最大限度地减少泄漏表达。对于慢病毒诱导基因表达载体系统,与多合一载体系统相比,双载体系统在四环素存在下可实现更高水平的转基因诱导。多合一载体由两个连续的表达盒组成:由TRE启动子驱动的GOI和由普遍存在或组织特异性启动子驱动的tTS / rtTA基因。对于慢病毒载体,不建议在每个表达盒的LTR之间放置内部聚腺苷酸化信号,因为这会抑制病毒包装。取而代之的是,单个聚腺苷酸化信号被放置在3'LTR中。因此,来自上游TRE启动子的转录通常通过下游启动子和ORF(tTS / rtTA基因)持续到上游ORF末端。这通常导致下游tTS/rtTA表达的部分抑制,从而阻止在四环素存在下有效诱导基因表达。因此,我们建议将靶细胞与携带TRE驱动的GOI的慢病毒和表达tTS / rtTA盒的慢病毒共转导,以实现最佳的诱导效率。
根据设计,慢病毒载体缺乏病毒包装和转导所需的基因(这些基因由病毒包装过程中使用的辅助质粒携带)。因此,由慢病毒载体产生的病毒具有复制不全的重要安全特征(这意味着它们可以转导靶细胞但不能在其中复制)。
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, 5' LTR-ΔU3 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 5'LTR-ΔU3 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 import of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.
Promoter: The promoter driving your gene of interest is placed here. Users can select between either the 2nd generation (TRE) or the 3rd generation (TRE3G) Tetracycline-responsive element promoter.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
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 gene promoter. It drives the ubiquitous expression 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.
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 (since the 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 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.
风险提示:丁香通仅作为第三方平台,为商家信息发布提供平台空间。用户咨询产品时请注意保护个人信息及财产安全,合理判断,谨慎选购商品,商家和用户对交易行为负责。对于医疗器械类产品,请先查证核实企业经营资质和医疗器械产品注册证情况。
有关该矢量系统的更多信息,请参阅以下论文。
引用 | 主题 |
---|---|
J 维罗尔。72:8463 (1998) | 第三代慢病毒载体 |
纳特·普罗托克。1:241 (2006) | 慢病毒载体的生产和纯化 |
科学。268:1766-9 (1995) | rtTA的发展。 |
基因医学杂志 1:4-12 (1999) | tTS的发展。 |