This vector system is designed for efficient analysis of mammalian cis-regulatory elements in vitro. Typically, a putative enhancer 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 enhancer elements, determining tissue- and spatial-specificity of enhancers, comparing enhancer 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.
|Genes Dev. 32:202 (2018)||Review on the prediction and identification of enhancers|
|Cell Stem Cell. 23:276 (2018)||Functional dissection of enhancers in human embryonic stem cells|
Our vector is based on a regular plasmid system. The putative enhancer to be tested is placed immediately upstream of a minimal promoter, which controls the expression of a downstream reporter gene. An active enhancer would stimulate the minimal promoter, driving reporter gene expression. In the absence of enhancer activity, the minimal promoter has very weak basal activity, and therefore produces 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 enhancer activity in vitro.
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 enhancer 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 enhancer activity in vitro.
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.
Enhancer: Your enhancer of interest is placed here.
Minimal promoter: A user-selected minimal promoter sequence is placed here. This will drive transcription of the downstream reporter if an enhancer element is present to activate it. In the absence of such enhancer activity, the minimal promoter will be almost completely inactive.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
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 enhancer 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.