质粒miR30-shRNA敲低载体

概述

质粒miR30 shRNA敲低载体系统,可在多种哺乳类细胞中进行高效瞬时靶基因敲低表达。该系统利用常规的质粒转染方式,将一个多顺反子表达框转入哺乳动物细胞中,该表达框包含一个或者多个基于miR30的靶向目的基因的shRNA(shRNAmiR)和一个用户选择的ORF。通过内源性胞内micro-RNA途径的处理,shRNAmiR转录本被加工成成熟的shRNAs,从而促使靶基因mRNAs的降解。

通过常规的转染方式将质粒载体递送到哺乳动物细胞中,是生物医药研究中使用最广泛的方式之一。尽管在过去的几年里,一些复杂的基因递送系统得到了进一步的开发与应用,例如慢病毒载体、腺病毒载体、腺相关病毒载体以及piggyBac载体等,传统的质粒转染仍然是许多实验室中基因递送的主要方式,这主要是归功于其在众多不同的细胞类型中技术操作简易和递送效率较好。使用质粒载体进行转染的关键特点就在于其瞬时表达,仅有非常少数的细胞会将质粒稳定整合到基因组中(通常<1%)。

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。

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
亮点

我们常规的miR30-shRNA敲低质粒载体利用一个优化的micro-RNA系统瞬时敲低目的基因,并且经过优化后,在大肠杆菌中具有较高的拷贝数以及高效的转染效率。该载体转染细胞后,可以通过用户选择的标记基因表达进行筛选和/或可视化。用户选择的启动子驱动多顺反子表达框,该表达框包含用户选择的ORF和一个或者多个经优化的、基于miR30序列的shRNAmiRs,用于介导有效的shRNA加工和靶基因的敲低。

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ORF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。

技术简单: 在技术上,通过常规的转染方式直接将质粒递送到细胞中,远比需要进行病毒包装的病毒载体简单的多。

高水平表达: 传统的质粒转染通常在细胞中拷贝数非常高(每个细胞中最高可达几千拷贝)。随之带来载体所携带的基因的高水平表达。

不足之处

载体DNA非整合: 常规质粒在细胞里主要以游离DNA状态存在,所以常规质粒转染也称为瞬时转染。因此,使用常规的miR30-shRNA敲低质粒载体进行的靶基因敲低通常也是瞬时的,因此该载体不适用于需要长期分析敲低表型的应用。不过,质粒DNA也可以以非常低的概率永久整合到宿主基因组中(每10^2~10^6个细胞可能会有一个细胞的基因组被整合,整合效率取决于细胞类型)。如果将携带了药物抗性基因或者荧光标记基因的载体转到细胞中,在经过扩大和传代培养后,可以使用药物筛选或细胞分选来获得稳定整合了该载体的细胞。

可转染的细胞类型有限: 质粒转染效率在不同细胞类型之间差异很大。非分裂细胞比分裂细胞更难转染,原代细胞比永生化细胞系更难转染。一些重要的细胞类型,如神经元细胞和胰腺β细胞,转染难度更是众所周知。此外,质粒转染主要局限于体外应用,而很少运用到体内实验。

基因递送的不均一性: 尽管一次成功的转染可以在单个细胞里产生非常高的拷贝数,但这在不同的细胞类型中表现出高度的不均一性,在一些细胞中,质粒拷贝数非常高,而在另外一些细胞却是低拷贝甚至没有。这不同于病毒载体转导,后者在细胞中的基因递送相对均一。

载体关键元件

Single miR30-shRNA regular plasmid shRNA knockdown vector

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.

SV40 late pA: Simian virus 40 late polyadenylation signal. Facilitates transcription termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.

CMV promoter: Human cytomegalovirus immediate early enhancer/promoter. This 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.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

Multiple miR30-shRNA regular plasmid shRNA knockdown vector

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.

SV40 late pA: Simian virus 40 late polyadenylation signal. Facilitates transcription termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.

CMV promoter: Human cytomegalovirus immediate early enhancer/promoter. This 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.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.