Tol2 miR30-Based shRNA Knockdown Vector

Overview

The Tol2 miR30-based shRNA knockdown vector system is a simple and efficient method for stably knocking down expression of target gene(s) in mammalian cells. This transposon-based system utilizes plasmid transfection (rather than viral transduction) to permanently integrate a polycistronic expression cassette consisting of one or more miR30-based shRNAs (shRNAmiR) targeting gene(s) of interest and a user-selected ORF into the host cell genome. The shRNAmiR transcript is processed by endogenous, cellular micro-RNA pathways to produce mature shRNAs, which facilitate degradation of target gene mRNAs. The permanent nature of knockdown by the Tol2 system has several major advantages over transient knockdown by synthetic siRNA (see Advantages section below).

The Tol2 miR30-based shRNA knockdown vector system contains two vectors, both engineered as E. coli plasmids. One vector, referred to as the helper plasmid, encodes the transposase. The other vector, referred to as the transposon plasmid, contains two terminal repeats (TRs) bracketing the region to be transposed, which includes the polycistronic shRNAmiR and ORF expression cassette. When the helper and transposon plasmids are co-transfected into target cells, the transposase produced from the helper plasmid recognizes the two TRs on the transposon, and inserts the flanked region including the two TRs into the host genome. Insertion occurs without any significant bias with respect to insertion site sequence. This is unlike transposon systems which have specific target consensus sites. For example, piggyBac transposons typically inserts at sites containing the sequence TTAA.

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). 

Tol2 is a class II transposon, meaning that it moves in a cut-and-paste manner, hopping from place to place without leaving copies behind. (In contrast, class I transposons move in a copy-and-paste manner.) Tol2 integrates as a single copy through a cut-and-paste mechanism. At each insertion site, the Tol2 transposase creates an 8 bp duplication, resulting in identical 8 bp direct repeats flanking each transposon integration site in the genome.

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
Genome Biol. 8(Suppl 1): S7 (2007) Review of Tol2 vectors
Genetics 174: 639–649 (2006) Identification of minimal sequences for Tol2 transposable elements
PLoS Genetics 2: e169 (2006) Large cargo-capacity transposition with a minimal Tol2 transposon

Highlights

Our Tol2 miR30-based shRNA knockdown vector incorporates an optimized micro-RNA system for knockdown of target gene(s).  This vector along with the helper plasmid are optimized for high copy number replication in E. coli, efficient, transfection into a wide range of target cells, and high-level expression of the transgene carried on the vector. 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.

Permanent integration and knockdown: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transfection of mammalian cells with the Tol2 transposon plasmid along with the helper plasmid can deliver DNA sequences carried on the transposon permanently into host cells due to the integration of the transposon into the host genome. As a result, the knockdown of the target gene(s) achieved with this vector is stable and permanent. This can be an important advantage for many experimental goals. It allows long-term analysis of the knockdown phenotype. It facilitates the isolation of clones having different levels of knockdown and/or different phenotypes. When the knockdown vector carries a fluorescence marker such as EGFP, it also allows cells with different amounts of transposon integration (and hence potentially different levels of knockdown) to be isolated by flow sorting cells with different fluorescence intensity.

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.

Safety: Conventional transfection does not have the safety concerns which are often associated with viral vectors.

Disadvantages

Limited cell type range: The delivery of Tol2 vectors into cells relies on transfection. The efficiency of 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. These issues limit the use of the Tol2 system.

Key components

Single miR30-shRNA Tol2 shRNA knockdown vector

5' ITR: Tol2 5' terminal repeat. When a DNA sequence is flanked by two ITRs, the Tol2 transposase 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.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.

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 Tol2 shRNA knockdown vector

5' ITR: Tol2 5' terminal repeat. When a DNA sequence is flanked by two ITRs, the Tol2 transposase 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.

SV40 late pA: Simian virus 40 late polyadenylation signal. It facilitates transcriptional termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.

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.

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