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载体构建质粒DNA制备
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mRNA基因递送解决方案
CRISPR基因编辑解决方案
shRNA基因敲低解决方案
LEX条件性表达慢病毒载体结合了载体家的高效的第三代慢病毒载体系统和Cre响应的FLEX条件性基因表达载体,可以帮助您实现在多种多样的哺乳动物细胞中以慢病毒转导Cre响应的FLEX基因表达调控开关。FLEX Cre-Switch系统的两个Lox-变体重组位点之间包含一对反向平行的ORF,在Cre重组酶的调控下ORF上的两个目的基因编码方向反转,从而让一个目的基因从激活表达变成沉默,另一个目的基因从沉默变成激活表达。
FLEX Cre-switch系统由两对异型Lox-变体重组位点组成,其中野生型序列称为LoxP,突变体称为Lox2272,分别位于两个基因ORF的两侧。 两个ORF相互反向,其中一个ORF具有正确的读码方向(相对于启动子)。两种Lox-变体都可被Cre识别,但只有相同的Lox位点对可以彼此重组,与其他Lox-变体不能进行重组。LoxP和Lox2272位点以交替方式位于两个ORF两端,每对位点互为反向。Cre重组酶不存在的情况时,第一个ORF由于其编码方向与启动子方向相同,可以在客户定制的启动子驱动下正常表达,而第二个ORF由于其方向相反则无法正常表达。在Cre的存在下,LoxP和Lox2272位点对分别重组,导致两个ORF方向反转,且其中的一对重组位点将具有相同的方向,Cre会介导产生正向重组切割,继而分别留下一个不同的重组位点。 ORF方向的反转,使得第二个OR可以在启动子的驱动下正常表达,而第一个ORF将不表达。 由于ORF侧翼是两个不同的Lox-变体位点,所以即使存在Cre也不会进一步发生重组。
慢病毒载体首先以质粒的方式构建并使用E. coli扩增,然后与多个辅助质粒一同转染至包装细胞。载体上的两个LTR区域之间的DNA序列将被转录成RNA,而辅助质粒表达的病毒蛋白进一步与这些RNA组装形成完整的病毒颗粒。有活性的病毒颗粒被释放到上清液中。病毒上清液可直接转染或者浓缩后转染细胞。
当慢病毒感染靶细胞时,病毒RNA被反转录成DNA,然后永久性整合到宿主基因组。位于两个LTR区域之间的载体组分永久整合到宿主基因组。对于上述的FLEX介导的Cre-Switch条件性表达慢病毒载体,FLEX Cre-Switch调控的两个反向平行的ORF位于两个LTR之间的区域。这两个ORF也被永久整合到宿主基因组当中,并在Cre调控下使得其中一个ORF获得激活表达,另一个则表达失活。
关于该载体系统的更多信息,请参考以下文献。
参考文献 | 主题 |
---|---|
J Virol. 72:8463 (1998) | The 3rd generation lentivirus vectors |
Nat Protoc. 1:241 (2006) | Production and purification of lentiviral vectors |
Gene. 216:55 (1998) | Characterization of LoxP mutants, including Lox2272 |
Nat Biotechnol. 21:562 (2003) | Development of the FLEX switch system |
J Neurosci. 28:7025 (2008) | Application of a FLEX switch system |
FLEX Cre-Switch条件性表达慢病毒载体专为在哺乳动物细胞和动物体内实现两个ORF可切换的基因表达而设计。目的基因表达受到用户自定义的启动子调节,并在Cre重组酶共表达的情况下沉默一个目的基因,并激活另一个目的基因表达。
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。
基因激活失活可控:两个ORF的方向彼此相反确保当表达正向方向的ORF时,反向的ORF被抑制,可防止基因发生泄漏表达。
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。
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。
安全性高: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。
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。
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.
Lox2272: Recombination site for Cre recombinase. Mutated Lox site with two base substitutions of wild type LoxP. Incompatible with LoxP sites. When Cre is present, the LoxP and LoxP2272 sites will be cut and recombine with compatible sites.
LoxP: Recombination site for Cre recombinase. Incompatible with Lox2272 sites. When Cre is present, the LoxP and Lox2272 sites will be cut and recombine with compatible sites.
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 #1: The open reading frame of a gene of interest is placed here, in a sense orientation. This gene can be expressed without Cre-mediated recombination.
ORF #2: The open reading frame of a gene of interest is placed here, in an antisense orientation. This gene can only be expressed after Cre-mediated recombination.
WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. It enhances transcriptional termination in the 3' LTR during viral RNA transcription, which leads to higher levels of functional viral RNA in packaging cells and hence greater viral titer. It also enhances transcriptional termination during the transcription of the user's gene of interest on the vector, leading to their higher expression levels.
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.