Regular Plasmid Promoter Testing Vector (for In Vivo Promoter Testing)

Overview

This vector system is designed for efficient analysis of mammalian promoters in mouse models. 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.

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
J Biol Chem. 273:10530 (1998) Analysis of promoter activity in vivo using a lacZ reporter plasmid

Highlights

Our vector is based on a regular plasmid system. The putative promoter to be tested is placed immediately upstream of a LacZ reporter gene. 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

Easy generation of transgenic animals: The construct can be readily used to make transgenic embryos or live mice with high efficiency by conventional pronuclear injection.

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.

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.

Very large cargo space: Our vector can accommodate ~30 kb of total DNA. This allows testing of large putative promoter sequences.

Disadvantages

Random integration into the host genome: When the vector is used to make transgenic mice by pronuclear injection, one or more copies of the vector can integrate randomly in the host genome. Neighboring genomic sequence at the integration site, coupled with copy number variation and varying degrees of chewing back of the integrated fragment, could influence the level and specificity of reporter gene expression. To overcome this, multiple transgenic lines are generally needed for a given construct, so as to identify the common expression pattern shared among the multiple lines, which is likely to be the true pattern rendered by the promoter.

Key components

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.

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.