PiggyBac Promoter Testing Vector (for In Vivo Promoter Testing)

Overview

This vector system is designed for efficient analysis of mammalian promoters in mouse models utilizing the piggyBac transposon system. Typically, a putative promoter of interest is cloned into this vector, upstream of a LacZ reporter gene 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 promoter activity. This vector system is useful for identifying promoter elements, determining tissue-specificity of promoters, comparing promoter 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 promoter 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.

References Topic
Comput Chem. 23:191 (1999) Review on eukaryotic promoter prediction
Mol Cell Biochem. 354:301 (2011) Review of the piggyBac systems
Cell. 122:473 (2005) Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice

Highlights

Our vector is based on the piggyBac transposon system. The putative promoter to be tested is placed immediately upstream of the LacZ reporter. While an active promoter would drive the expression of the downstream LacZ gene, in the absence of promoter activity there will little or no 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 promoter activity in situ.

Advantages

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 promoter 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 promoter sequences.

Disadvantages

Requires helper plasmid: Unlike the regular plasmid promoter-testing vector, the piggyBac vector system requires the use of two plasmids, a helper plasmid and a transposon plasmid. For effective insertion of the promoter sequence into the host genome, both plasmids must be present in the same cells.

Key components

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.

Promoter: Your promoter of interest is placed here.

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.

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