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

Overview

This vector system is designed for efficient analysis of mammalian promoters in vitro. Typically, a putative promoter of interest is cloned into this vector, and the resulting construct is used to transfect mammalian cell lines of interest. Expression of a downstream fluorescent or chemiluminescent reporter can then be used as a readout of enhancer activity.

This vector system is useful for identifying promoter elements, determining tissue-specificity of promoters, comparing promoter variants, and many other applications.

This vector can be introduced into mammalian cells by conventional transfection. Delivering plasmid vectors into mammalian cells by conventional transfection is one of the most widely used procedures in biomedical research. While several sophisticated gene delivery vector systems have been developed over the years such as lentiviral vectors, adenovirus vectors, AAV vectors and piggyBac, conventional plasmid transfection remains the workhorse of gene delivery in many labs. This is largely due to its technical simplicity as well as good efficiency in a wide range of cell types. A key feature of transfection with regular plasmid vectors is that it is transient, with only a very low fraction of cells stably integrating the plasmid in the genome (typically less than 1%).

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
Methods Mol Biol. 977:79 (2013) Characterization of a dual-luciferase reporter system for promoter analysis

Highlights

Our vector is based on a regular plasmid system. The putative promoter to be tested is placed immediately upstream of a reporter gene. While an active promoter would drive the expression of the downstream reporter gene, in the absence of promoter activity there will little or no reporter gene expression. A visually detectable bright fluorescent protein (such as TurboGFP) or a chemiluminescent protein (such as luciferase) is used as the reporter, which allows highly sensitive detection of promoter activity in vitro.

Advantages

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.

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

Simple and sensitive readout: A visually detectable bright fluorescent protein (such as TurboGFP) or a chemiluminescent protein (such as luciferase) is used as the reporter, resulting in highly sensitive readout of promoter activity in vitro.

Disadvantages

Limited cell type range: The efficiency of plasmid 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.

Key components

Promoter: Your promoter of interest is placed here.

Reporter: A visually detectable bright fluorescent protein gene (such as TurboGFP) or a chemiluminescent protein gene (such as luciferase). This allows highly sensitive detection of promoter activity in vitro.

SV40 late pA: Simian virus 40 late 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.

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