Regular Plasmid shRNA Knockdown Vector

Overview

Our regular plasmid shRNA knockdown vector system is a simple and efficient method for transiently knocking down expression of a target gene in a wide variety of cell types. This system utilizes conventional plasmid transfection to introduce an shRNA expression cassette into mammalian cells for knocking down a gene of interest. The shRNA is expressed from the human U6 promoter, leading to degradation of the target gene mRNA.

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%). 

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

References Topic
Methods Mol Biol. 629:141 (2010) Review on design and delivery of shRNAs
Cancer Gene Ther. 16:807 (2009) Comparison of siRNA and shRNA off target effects
Mol Cell Biol. 24:2614 (2004) Regular plasmid-mediated expression of shRNAs

Highlights

Our vector 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.

Advantages

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.

Ability to add selection markers: Our regular plasmid shRNA knockdown vector includes the option for users to add marker genes such as a drug selection gene or a visually detectable fluorescence gene. This allows cells transfected with the vector to be selected and/or visualized and therefore provides a distinct advantage over transient knockdown by synthetic siRNAs where no markers can be incorporated.

Reduced off-target effects: shRNAs have been seen to have reduced off-target effects compared to synthetic siRNAs since a much higher concentration of siRNA is required to achieve comparable levels of knockdown obtained with an shRNA construct, therefore potentially leading to increased off-target effects. Additionally, unlike shRNAs which are transcribed in the nucleus, siRNAs remain in the cytoplasm and are susceptible to degradation which could have further undesirable targeting effects.

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 regular plasmid shRNA vector can knock down genes of interest transiently in mammalian cells. 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

U6 Promoter: Drives expression of the shRNA. This is the promoter of the human U6 snRNA gene, an RNA polymerase III promoter which efficiently expresses short RNAs.

Sense, Antisense: These sequences are derived from your target sequences and are transcribed to form the stem portion of the “hairpin” structure of the shRNA.

Loop: This optimized sequence is transcribed to form the loop portion of the shRNA “hairpin” structure.

Terminator: Terminates transcription of the shRNA.

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression of the downstream marker gene. 

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.

Marker: A drug selection gene (such as neomycin resistance) or a visually detectable gene (such as EGFP). This allows cells transfected with the vector to be selected and/or visualized.

SV40 late pA: Simian virus 40 late 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.