载体指南
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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
我们的miR30-shRNA干扰AAV载体是一个能在体内或者体外进行目的基因敲低的高效病毒系统。由于AAV病毒的低免疫原性和细胞毒性,使得AAV成为许多动物研究的非常理想工具。该载体利用AAV介导的方式递送包含一个或者多个基于miR30的靶向目的基因的shRNAs(shRNAmiR)和一个用户选择的ORF的多顺反子表达框,该载体不整合到宿主基因组中,而是以游离DNA形式存在。shRNAmiR转录本通过内源的、细胞micro-RNA途径加工成成熟的shRNAs,以促进靶目的基因mRNAs的降解。
miR30-shRNA干扰AAV载体首先在大肠杆菌中构建成质粒,包含一个或者多个靶向目的基因的shRNAmiRs和一个用户选择的ORF的多顺反子表达框被克隆到两个ITRs之间。随后,与辅助质粒一起转染进入包装细胞中,两个ITRs之间的载体区域被包装进活病毒。两个ITRs之间的shRNAmiR表达框连同其他的病毒基因组一起转导到靶细胞中。
野生型AAV基因组是一条线性单链DNA(ssDNA),并含有两个ITRs区域在基因组的两端形成发夹结构,因此被称为ssAAV。为了能在宿主细胞中表达ssAAV载体携带的基因,ssDNA基因组首先需要通过两个途径转变成双链DNA(dsDNA):1)以ssDNA基因组作为模板、3' ITR作为引物位点,利用宿主细胞DNA聚合酶机制合成第二链DNA;2)ssAAV基因组的正义链和负义链之间形成分子内的dsDNA结构。两种途径中前者是形成dsDNA的主要方式。
AAV的DNA基因组在宿主细胞核中会形成游离形式的多联体。在非分裂细胞中,这些多联体在宿主细胞中保持活性。在分裂细胞中,AAV DNA会随着细胞分裂的稀释效应而丢失,这是因为游离的DNA不能伴随宿主细胞的DNA进行复制。AAV DNA也可随机合到宿主基因组上,但概率极小。AAV的这种特性应用于基因治疗是可取的,其中需要重点考量外源DNA整合到宿主基因组而造成的潜在致癌风险。
与利用RNA聚合酶III启动子(如U6启动子)的传统shRNA载体不同,miR-shRNA直接由标准的RNA聚合酶II类启动子驱动。这就可以使用组织特异性、诱导型或者不同强度的启动子,以实现采用常规U6启动子做不了的多种实验应用。
在miRNA-shRNA系统中,RNA聚合酶II启动子有效转录长转录本的能力比其他干扰载体系统有额外的优势。多个shRNAmiRs可以被转录为一个多顺反子,继而在细胞内被进一步加工成成熟的shRNAs。这允许去敲低使用一个转录本的多个基因或者靶向同一个基因的多个区域。因此,该载体可以用于表达一个或者多个shRNAmiRs。其次,在该载体系统中,用户选择的蛋白编码基因也可以与shRNAmiRs置于同一个多顺反子中。该ORF的表达可以用于直接检测shRNA的转录(如果ORF为标记基因),或者用于需要ORF和shRNAs共表达的其他目的。
AAV一个主要优势在于,在大多数情况下,可以在生物安全等级1(BSL1)的设施中处理AAV。这是因为AAV是复制缺陷型的,不会引起炎症反应和引发未知人类疾病。
人们已从自然界中鉴定出许多AAV病毒株,根据病毒表面衣壳蛋白的不同抗原将它们分成不同的血清型。不同血清型的病毒具有不同的组织嗜性(即感染组织特异性)。当我们的AAV载体使用不同的衣壳蛋白包装病毒时,则会赋予病毒不同的血清型。下表列出了不同的AAV血清型及对应的组织嗜性。
Serotype | Tissue tropism |
---|---|
AAV1 | Smooth muscle, skeletal muscle, CNS, brain, lung, retina, inner ear, pancreas, heart, liver |
AAV2 | Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes |
AAV3 | Smooth muscle, liver, lung |
AAV4 | CNS, retina, lung, kidney, heart |
AAV5 | Smooth muscle, CNS, brain, lung, retina, heart |
AAV6 | Smooth muscle, heart, lung, pancreas, adipose, liver |
AAV6.2 | Lung, liver, inner ear |
AAV7 | Smooth muscle, retina, CNS, brain, liver |
AAV8 | Smooth muscle, CNS, brain, retina, inner ear, liver, pancreas, heart, kidney, adipose |
AAV9 | Smooth muscle, skeletal muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney, adipose |
AAVrh10 | Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney |
AAV-DJ | Liver, heart, kidney, spleen |
AAV-DJ/8 | Liver, brain, spleen, kidney |
AAV-PHP.eB | CNS |
AAV-PHP.S | PNS |
AAV2-retro | Spinal nerves |
AAV2-QuadYF | Endothelial cell, retina |
AAV2.7m8 | Retina, inner ear |
Tissue type | Recommended AAV serotypes |
---|---|
Smooth muscle | AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10 |
Skeletal muscle | AAV1, AAV9 |
CNS | AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB |
PNS | AAV-PHP.S |
Brain | AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8 |
Retina | AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2-QuadYF, AAV2.7m8 |
Inner ear | AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8 |
Lung | AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10 |
Liver | AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8 |
Pancreas | AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10 |
Heart | AAV1, AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ |
Kidney | AAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8 |
Adipose | AAV6, AAV8, AAV9 |
Testes | AAV2, AAV9 |
Spleen | AAV-DJ, AAV-DJ/8 |
Spinal nerves | AAV2-retro |
Endothelial cells | AAV2-QuadYF |
更多关于该载体系统的信息,请参考下列文献。
References | Topic |
---|---|
Cell Rep. 5:1704 (2013) | An Optimized microRNA Backbone for Effective Single-Copy RNAi |
Methods Enzymol. 507:229-54 (2012) | Review of AAV virology and uses |
Curr Opin Pharmacol. 24:59-67 (2015) | AAV use in gene therapy, and serotype differences |
我们的miR30-shRNA干扰AAV载体包含了一个经过优化的用于敲低靶基因的micro-RNA系统,包装出来的病毒具有高滴度,能有效转导靶细胞,实现外源基因的高水平表达。该载体可用所有已知的衣壳蛋白血清型进行包装,包装出来的病毒具有非常高的转导效率,且具有极高的生物安全性。用户选择的启动子驱动包含一个或者多个基于miR30优化序列的shRNAmiRs和一个用户选择的ORF的多顺反子表达框表达,可以介导高效的shRNA加工和靶基因敲低。
启动子可选: 不同于使用RNA聚合酶 III 启动子(如U6启动子)的标准shRNA系统,基于miR30的shRNAs可以通过不同的RNA聚合酶II启动子进行转录,包括组织特异性启动子、诱导型启动子。
多shRNA共表达: 由于RNA聚合酶 II能有效地转录长RNAs,多个shRNAmiRs可以作为一个多顺反子被单个启动子表达。因此,该载体可以用于表达单个或者多个shRNAmiRs。
报告基因共表达: 用户可以选择感兴趣的基因或者报告基因ORF与 shRNAmiRs作为一个多顺反子进行共表达,用于直接监测shRNA转录。
安全性: AAV是可供选择的最安全的病毒载体,它是复制缺陷的,不会引起任何人类疾病。
对宿主基因组造成破坏的低风险性: 病毒转导到宿主细胞后,AAV载体以游离DNA的形式存在于细胞核中。AAV对宿主基因组的非整合特性使其减少了对宿主基因组的破坏而致癌的风险,可作为人类体内应用的理想载体。
高病毒滴度: 我们的AAV载体可以被包装成高滴度病毒,采用我们的AAV病毒载体体系包装AAV病毒,滴度可达>10^13 GC/ml。
广泛的亲嗜性: 针对不同来源的常用哺乳动物(如人、小鼠和大鼠)细胞和组织,将我们的AAV载体包装成对其具有亲和性的血清型,可轻易实现高效转导。但是,某些类型的细胞仍然难以转导(见下文不足之处),这与所用的血清型有关。
体内外实验均有效: 我们的AAV载体不仅能用于动物体内细胞转导,还具有体外细胞转导能力。
特定类型细胞转导困难: 通当利用合适的血清型进行包装时,我们的AAV载体系统可以转导很多不同类型的细胞,包括非分裂细胞。不同AAV血清型对不同类型的细胞具有不同的嗜性,针对某种特定类型的细胞需要特定血清型。
技术复杂: 使用AAV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。这些过程相对于常规质粒转染,技术难度更高,周期更长。不过,您可以在订购载体时直接选择我们的病毒包装服务。
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
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.
miR30-shRNA: This sequence is derived from your target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.
Regulatory element: Allows the user to add the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). WPRE enhances virus stability in packaging cells, leading to higher titer of packaged virus and enhances expression of transgenes.
BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.
3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.
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.
5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.
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.
miR30-shRNA #1: This sequence is derived from your first target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.
miR30-shRNA #2: This sequence is derived from your second target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.
miR30-shRNA #3: This sequence is derived from your third target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.
miR30-shRNA #4: This sequence is derived from your fourth target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.
Regulatory element: Allows the user to add the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). WPRE enhances virus stability in packaging cells, leading to higher titer of packaged virus and enhances expression of transgenes.
BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination and polyadenylation the upstream ORF and shRNAmiR polycistron.
3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.
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.