gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Regular Plasmid LacZ Reporter 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, 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.
For further information about this vector system, please refer to the papers below.
|Nature. 444:499-502 (2006)||Use of the vector systemtocarry out genome-wide testing of putative enhancers in the mouse|
|Development. 105:707-714 (1989)||Cloning of the Hsp68 minimal promoter|
Our vector is based on a regular plasmid system. The putative enhancer to be tested is placed immediately upstream of the Hsp68 minimal promoter (Hsp68_mini),which controls the expression of the downstream LacZ reporter. An active enhancer would stimulate the minimal promoter, driving LacZ expression. In the absence of enhancer activity, Hsp68_mini has very weak basal activity, and therefore produces 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 enhancer activity in situ.
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 enhancer activity.
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 enhancer.
Enhancer: Your enhancer of interest is placed here.
Hsp68 minimal promoter: The minimal promoter sequence from mouse Hsp68 (heat shock protein 68kDa). 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 will be almost completely inactive.
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.