gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
PiggyBac Enhancer Testing Vector (for In Vivo Enhancer Testing)
This vector system is designed for efficient analysis of mammalian enhancers in mouse models. Typically, a putative enhancer of interest is cloned into this vector, upstream of the mouse Hsp68 minimal promoter, and the resulting construct is used to make transgenic mice. Expression of the LacZ reporter in transgenic embryos or adult mice can then be used as a readout of enhancer activity. This vector system is useful for identifying enhancer elements, determining tissue-specificity of enhancers, comparing enhancer variants, lineage-tracing, and many other applications.
Our piggyBac transposon-based vector systems are highly effective for inserting foreign DNA into the host genome of mammalian cells. This system is technically simple, utilizing plasmids (rather than viral transduction) to permanently integrate your gene(s) of interest into the host genome.
The piggyBac 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 inverted terminal repeats (ITRs) bracketing the region to be transposed. The enhancer sequence of interest is cloned into this region. When the helper and transposon plasmids are co-injected or co-transfected into target cells, the transposase produced from the helper would recognize the two ITRs on the transposon, and insert the flanked region including the two ITRs into the host genome. Insertion typically occurs at host chromosomal sites that contain the TTAA sequence, which is duplicated on the two flanks of the integrated fragment after transposition.
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 introduced into host cells, it will get lost over time. With the loss of the helper plasmid, the integration of the transposon in the genome of host cells becomes permanent.
For further information about this vector system, please refer to the papers below.
|Nature. 444:499 (2006)||Genome-wide testing of putative enhancers in the mouse using a related system|
|Development. 105:707 (1989)||Cloning of the Hsp68 minimal promoter|
|Mol Cell Biochem. 354:301 (2011)||Review of the piggyBac systems|
Our vector is based on the piggyBac transposon system. The putative enhancer to be tested is placed immediately upstream of the mouse Hsp68 minimal promoter, which controls the expression of the downstream LacZ reporter. In the absence of enhancer activity, Hsp68 minimal promoter has very weak basal activity, and therefore produces little or no LacZ expression. An active enhancer would recruit transcription factors that support and enhance the transcriptional machinery, therefore stimulating the minimal promoter and activating LacZ expression. LacZ is used as the reporter because colorimetric staining of LacZ by X-gal in whole-mount embryos or tissue sections allows highly sensitive detection of enhancer activity in situ.
Simple and sensitive readout: When using LacZ as the reporter, X-gal staining produces a vivid blue product that is readily detectible even at low expression levels, resulting in very sensitive readout of enhancer activity.
Easy generation of transgenic animals: The construct can be readily used to generate transgenic embryos or live mice with high efficiency by conventional pronuclear injection.
Technical simplicity: Delivering plasmid vectors into cells by conventional transfection or injection is technically straightforward, and far easier than virus-based vectors which require the packaging of live virus.
Permanent integration of vector DNA: Use of conventional plasmids 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 or injection of 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, which allows long-term observation.
Very large cargo space: Our transposon vector can accommodate ~30 kb of total DNA. This allows testing of large putative enhancer sequences.
Requires helper plasmid: Unlike the regular plasmid enhancer-testing vector, the piggyBac vector system requires the use of two plasmids, a helper plasmid and a transposon plasmid. For effective insertion of the enhancer sequence into the host genome, both plasmids must be present in the same cells.
5' ITR: 5' inverted terminal repeat. When a DNA sequence is flanked by two ITRs, the piggyBac transposase can recognize them, and insert the flanked region including the two ITRs into the host genome.
Enhancer: Your enhancer of interest is cloned here.
Hsp68: The minimal promoter sequence from mouse Hsp68 (heat shock protein 68 kDa). This will drive transcription of the reporter if an enhancer element is present to activate it. In the absence of such enhancer activity, the minimal promoter only has basal activity.
LacZ: The beta-galactosidase reporter gene. The encoded enzyme converts the colorless and soluble X-gal to an intensely blue insoluble product that stains the cells in which LacZ is expressed.
SV40 early pA: Simian virus 40 early polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
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.