miR30-shRNA干扰PiggyBac载体

概述

miR30-shRNA干扰PiggyBac载体系统是一个可稳定干扰哺乳动物细胞靶基因表达的简单有效的方式。这种基于转座子的系统利用质粒转染(非病毒转导)将含有一个或多个基于miR30的靶向目的基因的shRNAs(shRNAmiR)和一个用户选择的ORF的多顺反子表达框永久地整合到宿主细胞基因组中。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。与合成siRNA的瞬时干扰相比,piggyBac系统在永久性干扰具有一些显著优势(见下文优势部分)。

miR30-shRNA干扰PiggyBac载体系统包含两个载体,均为工程化的大肠杆菌质粒,其中一个载体被称为辅助质粒,负责编码转座酶;另一个载体被称为转座子质粒,包含两个ITRs以及两个ITRs之间的被转座区域,shRNAmiR多顺反子和ORF表达框就在这个区域中。当辅助质粒和转座子质粒共转染靶细胞时,辅助质粒产生的转座酶会识别转座子上的两个TR元件,并将包含两个TRs的侧翼区域插入到宿主基因组中。转座插入通常发生在包含TTAA序列的宿主染色体位点上,并在转座子两侧出现TTAA重复序列。

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。 

PiggyBac属于II型转座子,通过“剪切—粘贴”的机制移动,从一个地方转座到另一个地方,而不遗留副本(相反,I型转座子是通过“复制-粘贴”的方式进行移动)。由于辅助质粒是通过瞬时转染进入宿主细胞的,因此会随着时间而逐渐丢失。随着辅助质粒的丢失,转座子将永久整合到在宿主细胞基因组中。当这些宿主细胞再次被辅助质粒转染,整合的转座子会再次通过“剪切—粘贴”的机制移动而不遗留痕迹。

关于该载体系统的更多信息,请参考以下文献。

ReferencesTopic
Cell Rep. 5:1704 (2013)An Optimized microRNA Backbone for Effective Single-Copy RNAi
Mol Cell Biochem. 354:301 (2011)Review of the piggyBac system
Cell. 122:473 (2005)Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice
亮点

我们的miR30-shRNA干扰PiggyBac载体系统包含了一个经过优化的用于敲低靶基因的micro-RNA系统,该PiggyBac转座子载体与辅助质粒是经过优化的,可在大肠杆菌中高拷贝复制,可在多种类型的靶细胞中进行高效转染、高效表达其携带的转基因。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。

永久性整合和干扰: 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该问题尤为显著。相反,使用PiggyBac转座子载体和辅助质粒一起转染到哺乳动物细胞中,由于转座子与宿主基因组的整合原理,可以将转座子上携带的DNA序列永久地整合到宿主细胞中。因此,该载体系统可进行稳定的和永久的靶基因干扰,这一特性对于许多实验目的而言具有重要优势,如可对培养细胞或活体干扰表型进行长期的干扰表型分析、有助于分离具有不同干扰水平和/或不同表型的克隆。当干扰载体携带荧光标记如EGFP时,还可通过流式分选具有不同荧光强度(荧光强度和整合数量有关,进而与干扰程度有关)的细胞。

可逆性: 如果再次使用辅助质粒转染携带PiggyBac shRNA转座子的细胞,可将表达shRNA的转座子从某些细胞的基因组中切除,而不留下任何痕迹。但是,这种情况只发生在一小部分细胞中。

技术简单: 通过常规转染的方法就可以直接把质粒递送进细胞中,相比起病毒载体需要进行病毒包装,过程更为简单。

安全性: 常规转染不会引起与病毒载体相关的安全性问题。

不足之处

可转染的宿主细胞类型有限: piggyBac载体依赖于转染方式进入细胞,在不同的细胞类型中转染效率差异非常大。非分裂细胞通常比分裂细胞更难转染,原代细胞比永生化细胞更难转染,一些重要的细胞类型转染难度更大,如神经元细胞和胰岛β细胞,转染的难度更是众所周知。此外,质粒转染主要局限于体外应用,而很少被应用于体内实验。这些问题都在一定程度上限制了piggyBac系统的应用。

载体关键元件

Single miR30-shRNA piggyBac shRNA knockdown vector

5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.

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.

rBG pA: Rabbit β-globin polyadenylation signal. Facilitates transcription termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of 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.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat.

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.

Multiple miR30-shRNA piggyBac shRNA knockdown vector

5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transpose can recognize them, and insert the flanked region including the two ITRs into the host genome.

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.

rBG pA: Rabbit β-globin polyadenylation signal. Facilitates transcription termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.

CMV promoter: Human cytomegalovirus immediate early promoter. It drives the ubiquitous expression of 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.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

3' ITR: 3' inverted terminal repeat.

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.