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Mouse Enhancer Screen Handbook and Methods

  1. Purpose
  2. Experimental Data
  3. Searching the Database
  4. Gallery
  5. Tutorial
  6. Reagent and Embryo Availability
  7. Citing the Enhancer Browser

1. Purpose

The goal of this project is to identify distant-acting transcriptional enhancers in the human and mouse genomes.  This is initially accomplished by a variety of computational and experimental analyses to identify putative enhancer elements followed by their testing in a transgenic mouse assay to validate their in vivo function and determine their activity patterns.

2. Experimental Data

2.1  Enhancer Candidate Identification

Most enhancer candidate sequences are identified by extreme evolutionary sequence conservation or by ChIP-seq.  Detailed information related to enhancer identification by extreme evolutionary conservation can be found in the following publications:

Note that previous releases of this web site included a “Computational Dataset” of evolutionarily conserved noncoding human sequences comprised of ~170,000 noncoding sequences that are highly conserved between humans and rodents. These datasets are no longer updated, but can now be obtained at:

Detailed information related to enhancer identification by ChIP-seq can be found in the following publications:

2.2  Transgenic Mouse Assay

In this section, we outline the experimental procedures that are used to perform these transgenic assays.

Primer design
PCR primers are designed to amplify candidate human or mouse enhancers.  Generally, conserved regions and ChIP-seq peaks are extended by several hundred basepairs in both directions in order to include flanking sequence that is required for enhancer activity but weakly conserved and/or adjacent to core coactivator binding sites.

In order to increase the throughput for reporter construct generation, we are exploiting the Gateway Recombination System (Invitrogen). Primers are 5' tailed with the required sequence (CACC) for cloning into Gateway vectors. PCR is performed on human genomic DNA (BD Biosciences).  PCR products are sequence verified and cloned into the standard Gateway entry vector (pENTR/D-TOPO vector, Invitrogen) using the manufacturer recommended protocol. We have generated a Gateway compatible reporter vector that contains a Gateway cassette and a Hsp68 promoter coupled to the LacZ reporter gene. Each Entry clone is transferred into the destination reporter vector using the LR recombination reaction and vector PCR and/or restriction enzyme analysis are used to confirm successful insert transfer.

Microinjection into fertilized eggs
Plasmid DNA is linearized with XhoI or HindIII, followed by purification on Montage PCR filter units and Micropure EZ columns (Millipore). The DNA is diluted with injection buffer (10 mM Tris, pH 7.5; 0.1 mM EDTA) to a final concentration of 1.5 to 2 ng/ul and used for pronuclear injections of FVB embryos in accordance with standard protocols approved by the Lawrence Berkeley National Laboratory.

Embryo harvesting and LacZ staining
Embryos are harvested at embryonic day 11.5 and dissected in cold PBS, followed by 30 min of incubation with 4% paraformaldehyde at 4oC. The embryos are washed three times for 30 min with wash buffer (2mM MgCl2; 0.01% deoxycholate; 0.02% NP-40; 100mM phosphate buffer, pH 7.3). Embryos are stained for 24 h at room temperature with freshly made staining solution (0.8mg/ml X-gal; 4mM potassium ferrocyanide; 4mM potassium ferricyanide; 20mM Tris, pH 7.5 in wash buffer) followed by 3 rinses in PBS and post-fixed in 4% paraformaldehyde. Yolk sacs are dissected from embryos and DNA prepared by boiling the tissue for 20 min in 75ml of solution 1 (25mM NaOH; 0.2mM EDTA), followed by neutralization with 75ml of solution 2 (40mM Tris-HCl). Yolk sac DNA is screened by PCR with LacZ primers (LacZ-F5'-TTTCCATGTTGCCACTCGC;LacZ-R5'-AACGGCTTGCCGTTCAGCA).

2.3  Annotation

Each positive developmental enhancer is annotated based on the observed spatial pattern of expression. These annotations are done by multiple curators in a group setting. To be defined as a POSITIVE enhancer, an element has to show reproducible expression in the same structure in at least three independent transgenic embryos. For each structure we provide the reproducibility of the observed pattern. Elements for which at least five transgenic embryos have been obtained, but no reproducible expression in any structure is observed in at least three different embryos are defined as NEGATIVE.

All embryos are preserved and, upon request, can be provided to outside investigators for their own examination and interpretation (see below)

A collection of schematic illustrations and selected examples for most annotated structures can be found on the Gallery page.

3. Searching the Database

3.1  General

As of May 2010, the Enhancer Browser contains transgenic reporter assay results for approximately 1300 elements.  These elements are either cloned from human or mouse genomic DNA.  The data can be most easily searched from the front page.  Searchable fields include:

-Gene names (of genes flanking tested enhancer elements)

-Gene accession numbers

-Locus link identifiers

-Genomic coordinates.  Note that genomic coordinates can be human (hg19) or mouse (mm9).  Not that if “both” is selected, the same coordinates in both species will be searched.

3.2  Advanced Search

On the advanced search page, additional search options are available.  These include:

-Searches for enhancers active in a particular tissue of interest (if multiple entries are selected, enhancers in any of the selected structures will be reported)

-Searches for only positive or only negative elements

-Searches restricted to only human or mouse enhancers

-Searches restricted to elements for which histological sections are available

3.3  Search Results

Search results are displayed as a list in which each row represents data for one construct.  In cases where both the human and the mouse orthologues were tested, two separate entries are provided.  Column “ID” contains the ID of each dataset; prefixes hs and mm refer to human or mouse DNA that was tested for enhancer activity, respectively.  Hyperlinked coordinates designate the genomic position in the species from which the enhancer was cloned; non-hyperlinked coordinates designate the orthologous region (where available) in the respective other species.  Hyperlinks “Human” and “Mouse” sort the table by element IDs in the respective species, hyperlinks “Coordinates” sort the table by coordinates in the respective species (intermixing original and orthologous coordinates in the respective other species).

The “Download Data” link enables bulk download of all search results as a single FASTA formatted plain text file that includes annotations and enhancer sequences.


blue mouse picturedesignates that a reproducible pattern was observed in the in vivo enhancer assay.

 white mouse picturedesignates that NO reproducible pattern was observed in the  in vivo enhancer assay.  Note that this annotation refers only to the single developmental timepoint that was tested in this screen (e11.5) and does not exclude the possibility that this region is a reproducible enhancer active at earlier or later timepoints in development.

sections available picture designates that sets of histological sections (in most cases of the brain) are available for at least one of the embryos in this dataset.

3.4  Data Set Pages

Drill-down pages are provided for each enhancer and can be accessed through the search results page.  Information on this page includes:

-Sequence coordinates

-Flanking genes

-Annotated expression patterns and details of reproducibility of each structure

-Photos of individual embryos (click on photo for a higher-resolution view)

-For selected elements, series of sections are available (typically coronal sections of the head).  Click on the single representative section to open the section browser.

-Sequence of the non-coding region that was tested

-Primer sequences that were used to amplify the candidate region from genomic DNA.  Note that for most constructs the reporter vector can be made available to outside investigators (see below).

-Overview of evolutionary sequence conservation of the tested region.

4. Gallery

The Gallery page contains schematic views and actual examples of enhancers active in most of the annotated structures for reference purposes.


An online tutorial about the Enhancer Browser and other VISTA comparative genomics resources is available from OpenHelix

6. Reagent and Embryo Availability

LacZ reporter vectors for most constructs, as well as archived (PFA-stored) transgenic embryos for selected elements can be made available to outside investigators upon request. Please inquire at Enhancer Support

7. Citing the Enhancer Browser

The following publication should be referenced for any analysis in which data from the VISTA Enhancer Browser was used:

Visel A, Minovitsky S, Dubchak I, Pennacchio LA (2007). VISTA Enhancer Browser-a database of tissue-specific human enhancers. Nucleic Acids Res 35:D88-92

When referring to specific datasets within the Enhancer Browser, please report the respective dataset ID, e.g. hs112 or mm23.

Last Updated May 2010.