Regular Plasmid miR30-Based shRNA Knockdown Vector

Overview

The regular plasmid miR30-based shRNA knockdown vector system is a highly efficient method for transiently knocking down expression of target gene(s) in a wide variety of mammalian cell types. This system utilizes conventional plasmid transfection for introducing a polycistronic expression cassette consisting of one or more miR30-based shRNAs (shRNAmiR) targeting gene(s) of interest and a user-selected ORF into mammalian cells. The shRNAmiR transcript is processed by endogenous, cellular micro-RNA pathways to produce mature shRNAs, which facilitate degradation of target gene mRNAs.

Delivering plasmid vectors into mammalian cells by conventional transfection is one of the most widely used procedures in biomedical research. While several sophisticated gene delivery vector systems have been developed over the years such as lentiviral vectors, adenovirus vectors, AAV vectors and piggyBac, conventional plasmid transfection remains the workhorse of gene delivery in many labs. This is largely due to its technical simplicity as well as good efficiency in a wide range of cell types. A key feature of transfection with regular plasmid vectors is that it is transient, with only a very low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).

Unlike conventional shRNA vectors, which utilize RNA polymerase III promoters such as U6, miRNA-based shRNA systems are placed under the control of standard RNA polymerase II promoters. This allows the use of tissue-specific, inducible, or variable-strength promoters, enabling a variety of experimental applications not possible with constitutive U6 promoters.

The ability of RNA polymerase II promoters to efficiently transcribe long transcripts in the miRNA-based shRNA systems also provides additional advantages relative to other knockdown vector systems. Multiple shRNAmiRs can be transcribed as a single polycistron, which is processed to form mature shRNAs within the cell. This allows knockdown of multiple genes or targeting of multiple regions within the same gene using a single transcript. As a result, this vector is available for expressing either single or multiple shRNAmiRs. Secondly, in this vector system, a user-selected protein coding gene is also positioned within the same polycistron as the shRNAmiRs. The expression of this ORF can be used to directly monitor shRNA transcription (if a marker ORF is used) or can be used for other purposes requiring co-expression of an ORF and shRNA(s).

For further information about this vector system, please refer to the papers below.

References Topic
Cell Rep. 5:1704 (2013) An Optimized microRNA Backbone for Effective Single-Copy RNAi

Highlights

Our regular plasmid miR30-based shRNA knockdown vector incorporates an optimized micro-RNA system for transient knockdown of target gene(s) and is optimized for high copy number replication in E. coli and high-efficiency transfection. Cells transfected with the vector can be selected and/or visualized based on marker gene expression as chosen by the user. A user-selected promoter drives expression of a polycistronic expression cassette containing a user-selected ORF and one or more shRNAmiRs with optimized miR30-based sequences to mediate efficient shRNA processing and target gene knockdown.

Advantages

Promoter choice: Unlike standard shRNA systems, which utilize RNA polymerase III promoters such as U6, miR30-based shRNAs can be transcribed by diverse RNA polymerase II promoters. This also enables the use of tissue-specific or inducible promoters.

Multiple shRNA co-expression: Because RNA polymerase II efficiently transcribes long RNAs, multiple shRNAmiRs can be expressed as a polycistron from a single promoter. Therefore, this vector is available for expressing either single or multiple shRNAmiRs.  

Co-expression of a reporter ORF: A user-selected gene of interest or reporter gene ORF is co-expressed with the shRNAmiRs, as a polycistron. This facilitates direct monitoring of shRNA transcription.

Technical simplicity: Delivering plasmid vectors into cells by conventional transfection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.

High-level expression: Conventional transfection of plasmids can often result in very high copy numbers in cells (up to several thousand copies per cell). This can lead to very high expression levels of the genes carried on the vector.

Disadvantages

Non-integration of vector DNA: Conventional transfection of plasmid vectors is also referred to as transient transfection because the vector stays mostly as episomal DNA in cells without integration. As a result, the knockdown of target genes achieved with the regular plasmid miR30-based shRNA knockdown vector is typically transient, therefore making the vector unsuitable for applications requiring long-term analysis of the knockdown phenotype. However, plasmid DNA can integrate permanently into the host genome at a very low frequency (one per 102 to 106 cells depending on cell type). If a drug resistance or fluorescence marker is incorporated into the plasmid, cells stably integrating the plasmid can be derived by drug selection or cell sorting after extended culture.

Limited cell type range: The efficiency of plasmid transfection can vary greatly from cell type to cell type. Non-dividing cells are often more difficult to transfect than dividing cells, and primary cells are often harder to transfect than immortalized cell lines. Some important cell types, such as neurons and pancreatic β cells, are notoriously difficult to transfect. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo.

Non-uniformity of gene delivery: Although a successful transfection can result in very high average copy number of the transfected plasmid vector per cell, this can be highly non-uniform. Some cells can carry many copies while others carry very few or none. This is unlike transduction by virus-based vectors which tends to result in relatively uniform gene delivery into cells.

Key components

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.

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