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基于腺病毒miR30的shRNA敲低载体系统是一种高效的病毒载体,用于敲低多种哺乳动物细胞类型中靶基因的表达。它利用腺病毒介导的多顺反子表达盒的递送,该盒由一个或多个基于miR30的shRNA(shRNAmiR)靶向感兴趣的基因和用户选择的ORF组成,其中载体保留为游离体DNA,而不整合到宿主基因组中。shRNAmiR转录本由内源性细胞微RNA途径处理以产生成熟的shRNA,从而促进靶基因mRNA的降解。它是体内首要选的基因敲低系统,常用于基因治疗和疫苗接种。
首先在大肠杆菌中构建基于腺病毒miR30的shRNA载体作为质粒。在两个倒置末端重复序列(ITR)之间克隆由一个或多个靶向目标基因的shRNAmiR和用户选择的ORF组成的多顺反子表达盒。然后将其转染到包装细胞中,其中ITR之间的载体区域被包装成活病毒。当病毒被添加到靶细胞中时,DNA货物被传递到细胞中,在那里它进入细胞核并保持为游离体DNA,而不整合到宿主基因组中。在载体构建过程中放置在两个ITR之间的任何基因都与病毒基因组的其余部分一起引入靶细胞。
与利用RNA聚合酶III启动子(如U6)的传统shRNA载体不同,基于miRNA的shRNA系统置于标准RNA聚合酶II启动子的控制之下。这允许使用组织特异性、诱导性或可变强度启动子,从而实现组成型 U6 启动子无法实现的各种实验应用。
相对于其他敲低载体系统,RNA聚合酶II启动子在基于miRNA的shRNA系统中有效转录长转录本的能力也提供了额外的优势。多个shRNAmiR可以转录为单个多顺子,其被加工成细胞内成熟的shRNA。这允许使用单个转录本敲低多个基因或靶向同一基因内的多个区域。因此,该载体可用于表达单个或多个shRNAmiR。其次,在该载体系统中,用户选择的蛋白质编码基因也位于与shRNAmiR相同的多顺子中。该ORF的表达可用于直接监测shRNA转录(如果使用标记ORF),或可用于需要ORF和shRNA共表达的其他目的。
根据设计,腺病毒载体缺乏E1A,E1B和E3基因(delta E1 + delta E3)。前两个是生产活病毒所必需的(这两个基因被设计到包装细胞的基因组中)。因此,由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)。
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.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
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.
TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. Facilitates transcription termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.
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.
Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.
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.
TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. Facilitates transcription termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.
ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.
3' ITR: 3' inverted terminal repeat.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.
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有关该矢量系统的更多信息,请参阅以下论文。
引用 | 主题 |
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细胞代表 5:1704 (2013) | 优化的microRNA骨架,实现有效的单拷贝RNAi |
美国国家科学院院刊 91:8802 (1994) | 第二代腺病毒载体 |
J 根维罗尔。36:59 (1977) | 腺病毒载体的包装细胞系 |
J 维罗尔。79:5437 (2005) | 293个细胞中复制能力腺病毒(RCA)的形成 |
吉恩·瑟。3:75 (1996) | 用于检测RCA的细胞系 |