miR30-shRNA干扰腺病毒载体

概述

基于miR30-shRNA的干扰腺病毒载体系统是一种能在多种哺乳动物细胞中高效敲低目的基因表达的病毒载体。该载体利用腺病毒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,该载体不会整合到宿主细胞的基因组中,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。该载体系统是体内首选的基因敲除系统,常被用于基因治疗和疫苗。

基于miR30-shRNA的腺病毒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shRNAmiR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。之后该质粒被转染到包装细胞中,载体上两个ITRs之间的区域被包装进活病毒中。病毒一经加入到靶细胞中,DNA内容物被递送到细胞中,并以游离DNA的形式存在于细胞核中,而不整合到宿主基因组中。在载体构建过程中,任何被插入到两个ITRs之间的基因片段都可以随其他的病毒基因组介导到靶细胞中。

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敲低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。

通过改造优化,我们的腺病毒载体缺少了E1A、E1B和E3基因(ΔE1+ΔE3),前两个基因是活病毒包装所需要的(这两个基因已被整合到包装细胞的基因组中)。因此,运用我们的该载体包装出来的病毒是复制缺陷型的(只能转导靶细胞而不能自我复制),大大提高了生物安全性。

更多关于该载体系统的信息,请参考下列文献。

ReferencesTopic
Cell Rep. 5:1704 (2013)An Optimized microRNA Backbone for Effective Single-Copy RNAi
Proc Natl Acad Sci U S A. 91:8802 (1994)The 2nd generation adenovirus vectors
J Gen Virol. 36:59 (1977)A packaging cell line for adenovirus vectors
J Virol. 79:5437 (2005)Replication-competent adenovirus (RCA) formation in 293 Cells
Gene Ther. 3:75 (1996)A cell line for testing RCA
亮点

我们的基于miR30-shRNA干扰腺病毒载体,由血清5型腺病毒(Ad5)衍生而来,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。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。

优势

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。

对宿主基因组造成破坏的低风险性: 在转导进入靶细胞后,腺病毒载体在细胞核保持着游离DNA的状态,可以降低宿主基因组被破坏而致癌的风险,使其成为人类体内实验的理想载体。

超高病毒滴度: 腺病毒可通过再次转导包装细胞而得到大量扩增,从而获得超高滴度病毒。这不同于像慢病毒、MMLV逆转录病毒或者腺相关病毒这一类病毒,它们均不能通过再次感染得到扩增。我们包装体系得到的腺病毒滴度可以达到>10^11IFU/ml。

广泛的亲嗜性: 我们的腺病毒载体可以广泛地转导如人、小鼠以及大鼠等常用的哺乳动物细胞,但是对于某些细胞类型则比较难转导(见下文不足之处)。

体内外实验均有效: 我们的腺病毒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。

安全性: 为了确保腺病毒载体的生物安全性,我们的腺病毒载体缺失了活病毒包装所必须的基因(这些基因被整合到包装细胞的基因组)。 所以,利用我们的腺病毒载体包装出来的病毒是复制缺陷型的,并且只有在包装细胞中才具有复制能力。

不足之处

载体DNA非整合型: 腺病毒基因组不能整合到靶细胞的基因组中,而以游离DNA形式存在,这会使得目标基因组随着时间推移而被逐渐丢失,尤其在分裂细胞中。 

特定类型细胞转导困难: 尽管我们的腺病毒载体可以转导包括非分裂细胞在内的多种不同类型的细胞,但是对某些特定类型的细胞难以进行有效的转导,比如内皮细胞、平滑肌细胞、分化的气道上皮细胞、外周血细胞、神经元和造血细胞等。

较强的免疫原性: 活腺病毒会在动物体内引起强烈的免疫反应,因此限制了其在体内的某些应用。

技术复杂: 使用腺病毒载体在包装细胞中获得活毒,然后进行病毒滴度测定。这些过程技术要求高,且周期长。

载体关键元件
Single miR30-shRNA adenovirus shRNA knockdown vector

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.

Multiple miR30-shRNA adenovirus shRNA knockdown vector

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.