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哺乳动物基因表达慢病毒载体

概述

慢病毒载体系统是一种能非常高效的把外源基因稳定整合到哺乳动物细胞中的载体工具。除了常规质粒转染外,目前该系统也是把外源基因转入哺乳动物细胞的最常用方法之一。由于具有目的基因和启动子选择的灵活性以及转染细胞类型的广泛性两大特点,使得慢病毒载体系统成为倍受欢迎的外源基因表达系统。

慢病毒载体来源于人类免疫缺陷病毒HIV,属于逆转录病毒家族。野生型慢病毒基因组是线性双正链RNA。

慢病毒重组载体构建完成后与辅助质粒一起转染进入包装细胞。在包装细胞中,位于两个长末端重复序列(LTR)之间的DNA片段会被转录成RNA,由辅助质粒表达的病毒蛋白将其包装形成病毒颗粒。包装后的活体病毒将会被释放到上清液中,可以直接收集或进一步浓缩病毒转染靶细胞。

当病毒转导靶细胞时,释放到宿主细胞中的病毒RNA借助逆转录酶逆转录成双链DNA,然后随机整合进宿主细胞的基因组中。在病毒载体中,位于两个LTR的DNA片段和病毒基因组都会稳定整合到靶细胞的基因组中。

通过改造优化,我们的慢病毒载体删除了与病毒包装和转导相关的基因(这些基因由辅助质粒进行表达,用于病毒包装过程),使产生的慢病毒颗粒是复制缺陷型的。即包装的病毒只具有转导靶细胞的能力,而无法在靶细胞中进行大量复制,因而具有很高的生物安全性。

关于慢病毒基因表达载体的更多信息,请参考以下文献。

参考文献主题
J Virol. 72:8463 (1998)The 3rd generation lentivirus vectors
J Virol. 72:9873 (1998)Self-inactivating lentivirus vectors
Science. 272:263 (1996)Transduction of non-dividing cells by lentivirus vectors
Curr Gene Ther. 5:387 (2005)Tropism of lentiviral vectors
J Virol. 77:4685 (2003)Impact of cPPT to lentivirus vector transduction
J Virol. 73:2886 (1999)WPRE enhances the expression of transgenes
Nat Protoc. 1:241 (2006)Production and purification of lentiviral vectors

亮点

我们目前采用的是第三代慢病毒包装载体系统。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。

优势

外源基因的稳定整合:常规质粒转染只能实现外源基因的瞬时表达,这种外源基因会随着宿主细胞的分裂而不断丢失,在快速分裂的细胞中显得尤为显著。相反的是,慢病毒转导的目的基因能稳定地整合到宿主细胞的染色体中 ,因而会随着宿主细胞的分裂而稳定遗传。

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。

灵活使用启动子:我们的慢病毒载体已经过优化,其5' LTR的启动子已进行了自失活。因此,客户可以灵活使用启动子来驱动目的基因的表达。这相对于只能依赖自身5' LTR启动子的MMLV载体来说是一个巨大的优势。

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。

不足之处

载体容量受限:野生型的慢病毒基因组大小约为9.2 kb,而在我们的慢病毒载体中,病毒包装和转导的必要元件约为2.8 kb,余下6.4 kb的空间容纳客户的目的序列。当病毒载体超过以上大小限制,病毒的包装滴度将会大大降低。我们的慢病毒载体除了可以插入靶基因的序列外,还可以插入启动子和筛选标记等载体元件。如果目的基因和这些载体元件长度超过了6.4 kb,病毒的产量有可能会明显下降。

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。

载体关键元件

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: The promoter driving your gene of interest is placed here.

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 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.

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.

Representative vector design
VB IDVector nameDescriptions
VB010000-9298rtf pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherryA 3rd generation mammalian gene expression lentiviral vector encoding EGFP and a puromycin resistance gene (linked by T2A) driven by a CMV promoter as well as mCherry under the control of an EF1A promoter.
VB900110-2722rff pLV[Exp]-EGFP:T2A:Puro-EF1A>hHBB[NM_000518.5]A lentiviral gene expression vector encoding EGFP and puromyocin resistance (linked by T2A) under the control of a CMV promoter as well as a human beta-globin gene driven by EF1A.