gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
PiggyBac shRNA Knockdown Vector
Our piggyBac shRNA Knockdown vector system is a simple and efficient method for stably knocking down expression of a target gene in a wide variety of cell types. This transposon-based system utilizes plasmid transfection (rather than viral transduction) to permanently integrate an shRNA expression cassette into the host cell genome. The shRNA is expressed from the human U6 promoter, leading to degradation of target gene mRNA. The permanent nature of knockdown by the piggyBac system has several major advantages over transient knockdown by synthetic siRNA (see Advantages section below).
The piggyBac shRNA knockdown 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 shRNA-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. PiggyBac 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.) Because the helper plasmid is only transiently transfected into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the shRNA-expressing transposon in the genome of host cells becomes permanent. If these cells are transfected with the helper plasmid again, the transposon could get excised from the genome of some cells, footprint free.
For further information about this vector system, please refer to the papers below.
|Mol Cell Biochem. 354:301 (2011)||Review|
|Cell. 122:473 (2005)||Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice|
Our piggyBac transposon vector along with the helper plasmid are optimized for high copy number replication in E. coli, and efficient transfection into a wide range of target cells. The human U6 promoter drives high-level, constitutive transcription of the shRNA in mammalian cells, while our optimized shRNA stem-loop sequences mediate efficient shRNA processing and target gene knockdown.
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 piggyBac 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. Additionally, the U6 promoter directs constitutive expression of the shRNA. For these reasons, the knockdown of the target gene 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.
Reversibility: If cells carrying a piggyBac shRNA transposon are transfected with the helper plasmid again, the transposon may be excised from the genome of some cells, footprint free, eliminating expression of the shRNA from those cells. However, this will occur in only a subset of cells.
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.
Limited cell type range: The delivery of piggyBac 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. Additionally, plasmid transfection is largely limited to in vitro applications and rarely used in vivo. These issues limit the use of the piggyBac system.
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.
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.
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.
rBG pA: Rabbit β-globin polyadenylation signal. It facilitates transcriptional termination of the upstream marker gene.
3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.
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.