The authors would like to acknowledge Dr. Bence Daniel, Matt Peloquin, Dr. Endre Barta, Dr. Balint L Balint and members of the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by grants from the Hungarian Scientific Research Fund (OTKA K100196 and K111941) and co-financed by the European Social Fund and the European Regional Development Fund and Hungarian Brain Research Program - Grant No. KTIA_13_NAP-A-I/9.

1. Enhancer Selection Based on Chip-seq Analysis
<ol>
	<li>Download the RXR ChIP-seq raw data fastq file (mm_ES_RXR_24h_ATRA.fastq.gz) from http://ngsdebftp.med.unideb.hu/bioinformatics/</li>
	<li>Download and extract the required BWA index file for the alignment (in our case: Mus_musculus_UCSC_mm10).(ftp://igenome:G3nom3s4u@ussd-ftp.illumina.com/Mus_musculus/UCSC/mm10/Mus_musculus_UCSC_mm10.tar.gz 
	NOTE: Visit to https://github.com/ahorvath/Bioinformatics_scripts for more information regarding the steps of bioinformatics analysis and to download the scripts used below.</li>
	<li>Align the example fastq file to mm10 genome (use the script: perform_alignment.sh). This will create a folder with a .bam file and statistics of the alignment. Aligned RXR ChIP-seq data (mm_ES_RXR_24h_ATRA.bam), and the corresponding index file (.bai) are available here: 
	http://ngsdebftp.med.unideb.hu/bioinformatics/ 
	NOTE: BWA is a software package that aligns relatively short sequences (e.g., ChIP-seq results) to a sequence database, such as the mouse reference genome (e.g., mm10) 13.</li>
	<li>Run the script (callpeaks.sh) for peak calling and de novo motif analysis. Use the .bam file as the input. The script is based on Homer findPeaks 14. 
	NOTE: The output files give us information about the total number of peaks, enrichment of the motifs, the percentage of the background, and the target sequences with motifs, etc. (Figure 1). Typically several motifs are listed in order of their significance.</li>
	<li>Remap the #1 ranked motif (NR half `AGGTCA`) (use the script: remap_motif.sh) 
	NOTE: As the result, the script generates a .bed file that will show which ChIP-peaks are covering genomic regions that contain the canonical binding site of the transcription factor of interest.</li>
	<li>Download and visualize results of the aligned RXR ChIP-seq reads (mm_ES_RXR_24h_ATRA.bam (also download the .bai)) and the AGGTCA motif occurrences (mm_ES_RXR_24h_ATRA_homerpeaks_motif1_mm10s_200_remaped_mbed.bed) by Integrative Genomics Viewer (IGV)15 to identify putative RAR/RXR binding sites (Figure 2 and Figure 3). 
	NOTE: Integration of various ChIP-seq data will help to more precisely predict good candidate enhancers16,17. Recent studies revealed that active enhancers are typically enriched for P300 and correlate with H3K4me1 and H3K27ac, and are located in open chromatin regions, thereby displaying DNase-I hypersensitivity is also recommended 18-21.</li>
	<li>Use the &#34;Define a region of interest&#34; in IGV 15 to obtain 200 - 400 bp sequence of the selected genomic region and use these sequences for subsequent primer designs.</li>
</ol>
2. Reporter Assay
NOTE: The luciferin/luciferase system is used as a very sensitive reporter assay for transcriptional regulation. Depending on the enhancer activity luciferase enzyme is produced that will catalyze the oxidation of luciferin to oxyluciferin resulting in bioluminescense, which can be detected. As a first step, identified putative enhancer sequences should be subcloned into a reporter vector (e.g., TK-Luciferase 22, pGL3 or NanoLuc).
<ol>
	<li>Primer Design
	<ol>
		<li>Design PCR primers for amplification of 200 - 400 bp of putative enhancer regions by Primer3 (available here: http://bioinfo.ut.ee/primer3/)23</li>
		<li>Copy-paste the genomic sequence from IGV that includes the putative binding site into Primer3 (see step 1.7). Set product size range to 250 - 300 bp in Primer3.</li>
		<li>Validate the PCR primers using the UCSC Genome Browser&#39;s In silico PCR tool (https://genome.ucsc.edu/cgi-bin/hgPcr)24 
		NOTE: This protocol in a later step uses HindIII and BamHI restriction enzymes for cloning. Check whether the PCR amplified sequence as predicted by the UCSC In-silico PCR tool will contain AAGCTT or GGATCC restriction sites (e.g., &#160;http://nc2.neb.com/NEBcutter2/).</li>
		<li>Obtain the required primers from a commercial vendor.</li>
	</ol>
	</li>
	<li>PCR Amplification of Enhancer Sequence from Genomic DNA
	<ol>
		<li>Purify template genomic DNA from primary cells (e.g., primary embryonic fibroblasts). Use commercial kit for gDNA isolation and follow the manufacturer&#39;s instructions. 
		NOTE: High quality of gDNA is essential for successful PCR amplification. Appropriate BAC clones can be used if PCR is not easy from gDNA.</li>
		<li>Prepare 50 &#181;l PCR reaction containing 100 ng gDNA, 5 &#181;l 10x buffer, 3 &#181;l MgSO4 (25 mM), 5 &#181;l dNTP (2 mM each), 1.5 &#181;l Fwd primer (10 &#181;M), 1.5 &#181;l Rev primer (10 &#181;M), 1 &#181;l DNA polymerase 
		NOTE: Use high-fidelity DNA polymerase with a low error rate for the PCR reactions.</li>
		<li>Set the PCR cycle (2 min 95 &#176;C, (20 sec 95 &#176;C, 10 sec 58 - 65 &#176;C, 18 sec 70 &#176;C) x25 repeat, 2 min 70 &#176;C, forever 4 &#176;C). Set annealing temperature with the consideration of the primer&#39;s predicted Tm values.</li>
		<li>Purify the PCR product with a commercial PCR purification kit and follow the manufacturer&#39;s instructions.</li>
		<li>Introduce the restriction sites for cloning by repeating the PCR with primers as used above, but adding overhangs on their 5&#39; ends (e.g., Fwd: 5&#39;-ATATAAGCTTxxxxxxxxxxxxxxxxxxxx-3&#39; (HindIII), Rev: 5&#39;-TATAGGATCCxxxxxxxxxxxxxxxxxxxx-3&#39; (BamHI)).</li>
		<li>Run 5 &#181;l of the PCR product on agarose gel to check for unspecific PCR products. 
		NOTE: If more bands are detected, run the entire PCR product and purify the appropriate band by a commercial gel extraction kit.</li>
	</ol>
	</li>
	<li>Restriction 
	NOTE: To clone insert upstream of TK promoter22 the purified PCR product and the vector (e.g., TK-Luc) should be digested with HindIII and BamHI.
	<ol>
		<li>Prepare a 50 &#181;l reaction mixture containing 1 &#181;g purified PCR product, 1 &#181;l HindIII (20 U/&#181;l), 1 &#181;l BamHI (20 U/&#181;l) and 5 &#181;l 10x buffer.</li>
		<li>Prepare a 250 &#181;l reaction mixture containing 5&#181;g vector (TK Luc, or alternative reporter vector), 5 &#181;l HindIII (20 U/&#181;l), 5 &#181;l BamHI (20 U/&#181;l), 5 &#181;l thermosensitive Alkaline Phosphatase (1 U/&#181;l) and 25 &#181;l 10x buffer. 
		NOTE: AP treatment of the vector greatly decreases the self-ligation of single-digested vector and thus the background.</li>
		<li>Incubate the reaction mixtures for 4 hr at 37 &#176;C, then purify the digested PCR products and the vector with a commercial PCR purification kit.</li>
	</ol>
	</li>
	<li>Ligation
	<ol>
		<li>Set up the reaction for ligation in PCR tubes including 2 &#181;l 10x T4 DNA ligase buffer, 10 ng TK-Luc vector, 50 ng insert (digested PCR product), 1 &#181;l T4 DNA ligase (400 U/&#181;l) and nuclease-free water up to 20 &#181;l. 
		NOTE: Prepare a negative control where the reaction does not contain insert.</li>
		<li>Gently mix the reaction by pipetting up and down, spin down briefly the PCR tubes using mini-centrifuge and then incubate at RT for 10 min.</li>
		<li>Heat inactivate at 65 &#176;C for 10 min then chill on ice and use 5 &#181;l of the product for transformation into 50 &#181;l competent cells (e.g., DH5&#945;).</li>
		<li>Use standard heat shock procedure to transform bacteria, pick up colonies and isolate plasmid DNA with a commercial kit. Validate all constructs by sequencing prior to the next steps.</li>
	</ol>
	</li>
	<li>Transfection of ES Cells
	<ol>
		<li>Use 48-well plates. Add 200 &#181;l 0.1% gelatin solution/well 30 min prior to cell plating.</li>
		<li>Plate embryonic stem (ES) cells a day before transfection. Use feeder-free ES cells and plate at a density of 3 x 104 cells per well in 250 &#181;l ES media/well.</li>
		<li>Prepare the plasmid mixes (each mix is calculated for 8 wells, 4 untreated and 4 retinoic acid-treated). Mix 1: 1,250 ng TK-Luc Empty (negative control), 950 ng &#946;-Galactosidase coding vector (&#946;Gal), Mix 2: 1,250 ng TK-Luc Hoxa1 enhancer (positive control), 950&#160;ng &#946;Gal, Mix 3: 1,250 ng TK-Luc PRMT8 enhancer (region of interest), 950 ng &#946;Gal. 
		NOTE: ES cells express the desired transcription factors (RAR/RXR). Leave wells untransfected to determine base values later during the luciferase and &#946;-galactosidase measurements.</li>
		<li>Add transfection quality reduced serum media to each plasmid mix up to 106 &#181;l total volume (enough for 8 wells).</li>
		<li>Add 4.5 &#181;l ES quality transfection reagent then mix carefully by pipetting 15 times up and down. Incubate the transfection mix for 10 min at RT.</li>
		<li>Add 13 &#181;l of the transfection mix to the cells per well, mix thoroughly by pipetting. Place the cells back into the incubator (37 &#176;C) O/N before ligand treatment.</li>
		<li>Remove media carefully by aspiration from cells and add 250 &#181;l fresh media/well containing 0.01 - 1 &#181;M all-trans retinoic acid (RA) as final concentration or DMSO as vehicle. Incubate cells for 24 - 48 hr. 
		NOTE: Retinoic acid is light sensitive.</li>
	</ol>
	</li>
	<li>Preparation of Cell Lysates
	<ol>
		<li>Dilute 5x Lysis Buffer (1.25 ml 0.5 M Tris pH = 7.8, 1 ml 1 M dithiothreitol (DTT) (in H2O), 10 ml 0.1 M EDTA pH = 8.0, 50 ml glycerol, 5 ml Triton X-100, sterile water up to 100 ml) to 1x using sterile water, add 20 &#181;l 1 M DTT/10 ml and equilibrate to RT before use. Prepare 10 ml for a 48-well plate on the day of the assay.</li>
		<li>Remove media carefully by aspiration from cells. Rinse cells once with 1x PBS. Add 200 &#181;l 1x Lysis Buffer/well directly to the cells.</li>
		<li>Shake for 2 hr at RT (chemical lysis). Freeze the cell lysates on the plate at -80 &#176;C (mechanical lysis). Lysates can be stored at -80 &#176;C for several days.</li>
		<li>Thaw the lysates. After complete thawing, transfer cell lysates to a 96-well plate using an electronic multichannel pipette. Transfer 80 &#181;l of the cell lysate to 96-well clear plate for &#946;-galactosidase assay and 40 &#181;l of the cell lysate to a white plate for luciferase measurement. Avoid forming any bubbles.</li>
	</ol>
	</li>
	<li>&#946;-galactosidase Assay 
	NOTE: Beta galactosidase or alternative second reporters should be used as an internal control to account for non-specific experimental variations.
	<ol>
		<li>Prepare &#946;-galactosidase substrate buffer: 80.0 g Na2HPO4&#8226;7H2O, 3.8 g NaH2PO4&#8226;H2O, 30.0 g KCl, 1.0 g MgSO4&#8226;7H2O, make up to 1,000 ml with sterile water. Adjust pH to 7.4. Filter Sterilize (0.2 micron) and store at 4 &#176;C</li>
		<li>Mix 1 ml &#946;-galactosidase substrate buffer with 4 mg ONPG (o-nitrophenyl-&#946;-D-galactosidase). Add 3.5 &#181;l of 2-Mercaptoethanol (&#946;ME) per 1 ml of buffer just before use. Add 100 &#181;l &#946;-galactosidase substrate buffer to 80 &#181;l cell lysate.</li>
		<li>Incubate the reactions at 37 &#176;C until a faint yellow color has developed. Read absorbance at OD 420 nm on a plate reader and export data. 
		NOTE: The time varies depending on the transfection efficiency, usually no longer than 30 min.</li>
	</ol>
	</li>
	<li>Luciferase Assay
	<ol>
		<li>Prepare 50 ml luciferin substrate reagent: 15.1 mg D-luciferin salt, 82.7 mg ATP salt, 185 mg MgSO4&#8226;7H2O, add to 50 ml polypropylene tube, then add 1.5 ml 1 M HEPES buffer, 48.5 ml ATP free water, vortex, make sure all compounds dissolve completely. Keep 5 - 10 ml aliquots at -80 &#176;C.</li>
		<li>Warm 5 ml luciferin substrate reagent to RT before starting. Pipette 100 &#181;l substrate reagent to each well of the white plate containing the 40 &#181;l of the cell lysate and measure the signal immediately using a luminescent counter machine.</li>
		<li>Obtain activities from at least three independent experiments in triplicate transfections.</li>
		<li>Calculate first the base normalized luciferase activity and base normalized &#946;-galactosidase values for each well by subtracting the average values measured for non-transfected cells from each value measured. Then calculate the ratio of luciferase/&#946;-galactosidase activity.</li>
	</ol>
	</li>
</ol>
3. Characterization of Enhancer RNA
NOTE: A more direct indicator of enhancer activity has emerged from recent genome-wide studies that identified many short non-coding RNAs, ranging in size from 50 to 2,000 nt, which are transcribed from enhancers, and are termed enhancer RNAs (eRNAs)16,25,26 (Figure 4). eRNA induction highly correlates with the induction of adjacent exon-coding genes. Thus, signal-dependent enhancer activity can be quantified in vivo by comparing eRNA production between various conditions by RT-qPCR.
<ol>
	<li>Guidelines for Primer Design for eRNA&#160;detection by RT-qPCR
	<ol>
		<li>Select genomic regions for eRNA measurements that are at least 1.5 - 2 kb away from annotated transcription start sites. 
		NOTE: Importantly, high levels of mRNA transcription across intragenic enhancers prevent accurate quantification of eRNA in the sense orientation, antisense eRNAs at intragenic enhancers are detectable and are similar in level to eRNAs at extragenic enhancers.</li>
		<li>As a general rule, use regions 200 - 1,000 bp from the center of the transcription factor binding site of interest for primer design either on the sense or anti-sense strand (Figure 4 and Figure 6). 
		NOTE: If GRO-seq, DNase-seq, H3K4me1, H3K4me3 or H3K27ac ChIP-seq data are available from the desired cell types and conditions it can be used for more accurate primer design. eRNAs are transcribed from putative enhancer regions characterized by high levels of H3K4me1 and me3. Expression of eRNAs positively correlates with the enrichment of activated enhancer histone mark H3K27ac.</li>
		<li>Design PCR primers for fluorescent dye-based RT-qPCR measurement of eRNAs by Primer3 (http://bioinfo.ut.ee/primer3/)23Generate anti-sense sequence (reverse complement of the sense strand) for primer design as per: http://www.bioinformatics.org/sms/rev_comp.html. Insert 200 - 300 bp of sense or anti-sense sequence for primer design.</li>
	</ol>
	</li>
	<li>Measurement of eRNA Transcription
	<ol>
		<li>Isolate total RNA from treated cells with commercial acidic phenol/chloroform-based extraction reagent according to the manufacturer&#39;s recommendations.</li>
		<li>Use DNaseI to treat isolated RNA prior to using them in reverse transcription reaction. Inactivate DNase according to the manufacturer&#39;s recommendation prior to reverse transcription.</li>
		<li>Measure RNA concentration after DNase I treatment and use 1,000 ng per RT reaction. Use high quality reverse transcriptase. 
		NOTE: As large number of eRNAs may not be polyadenylated, reverse transcription requires the use of random primers.</li>
		<li>Detect reverse transcribed eRNA by RT-qPCR using standard procedure. Measure a non-treatment-dependent mRNA for normalization (e.g., 36B4, Ppia, Gapdh, Actb).</li>
	</ol>
	</li>
</ol>

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Sox2+distal+enhancer+cluster+regulates+embryonic+stem+cell+differentiation+potential.">A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential.</a> Genes Dev. 28, 2699-2711 (2014).</li></ol>

The authors have nothing to disclose.

In recent years, advances in sequencing technology have allowed large-scale predictions of enhancers in many cell types and tissues 7-9. The workflow described above allows one to perform primary characterization of candidate enhancers chosen based on ChIP-seq data. The detailed steps and notes will help anyone to set up a routine enhancer validation in the lab.
The most critical step in the luciferase reporter assay is the transfection efficiency. It is recommended to include a GFP-construct in order to estimate the transfection efficiency in every experiment. Having fresh and good quality of plasmids can be critical and can significantly improve the transfection. The presented protocol can be adapted to any cellular model system. It is highly recommended to use the appropriate cell type for the characterization of tissue-specific enhancers. Thus, luciferase reporter assay requires that the cells chosen for the experiment should be efficiently transfectable. Alternatively, easily transfectable cells should be used where the transcription factor of interest is (over)expressed. This can be particularly important in case of some tissue-specific enhancers. 
In contrast, enhancer RNA can be easily used in any cell without such limitations to study enhancer activity in different conditions in vivo. Key parameters for efficient eRNA detection are listed above. Isolation of good quality, non-degraded RNA is essential. DNase treatment is very important in order to avoid the detection of genomic DNA contamination instead of the eRNA. As RNA is used for subsequent reverse transcription reaction to get cDNA, DNase should be appropriately inactivated. It is advisable to design primers for the detection of both sense and anti-sense eRNAs.
Carrying out the above described enhancer trap experiment (transfection of a construct containing an enhancer element and a luciferase-based reporter) can provide strong functional evidence for enhancer activity. However, the ultimate proof, which is rarely presented in research articles, would be genetic elimination of the designated enhancer and thus determination of its role in vivo or at least in a cell 28.
The above-described workflow does not give us information which gene is regulated by the validated enhancer. Further research could use 3C (Chromosome Conformation Capture) experiment that show whether the implicated enhancer is interacting with the promoter of the gene of interest.

Development of a multicellular organism requires precisely regulated expression of thousands of genes across developing tissues. Regulation of gene expression is accomplished in large part by enhancers. Enhancers are short non-coding DNA elements that can be bound with transcription factors (TFs) and act from a distance to activate transcription of a target gene1. Enhancers are generally cis-acting and most frequently found just upstream of the transcription start site (TSS), but recent studies also described examples where enhancers were found much further upstream, on the 3' of the gene or even within the introns and exons2. 
There are hundreds of thousands of potential enhancers in the vertebrate genomes1. Recent methods based on chromatin immunoprecipitation (ChIP) provide high-throughput data of the whole genome that can be used for enhancer analysis3-9. Though data obtained by ChIP-seq experiments greatly increases the likelihood to identify cell and tissue-specific enhancers, it is important to keep in mind that detected binding sites do not necessarily identify direct DNA binding and/or functional enhancers. Thus, further functional analysis of newly identified enhancers is indispensable. In this work, we present a basic three-step process of putative active enhancer identification and validation. This includes: 1) selection of putative transcription factor binding sites by bioinformatics analysis of ChIP-seq data, 2) cloning and validation of these regulatory sequences in reporter constructs, and 3) measurement of enhancer RNA (eRNA). 
Exposure of embryonic stem (ES) cells to retinoic acid (RA) is frequently used to promote neural differentiation of the pluripotent cells 10. RA exerts its effects by binding to RA receptors (RAR&#945;, &#946;, &#947;) and retinoid X receptors (RXR&#945;, &#946;, &#947;). RARs and RXRs in a form of heterodimer bind to DNA motifs called RA-response elements, that is typically arranged as direct repeats of AGGTCA sequence (called as half site) and regulate transcription. Ligand-treatment experiments allowed the identification of several retinoic acid regulated genes in ES cells 11,12. However, enhancer elements for many of these genes has not been described yet. To demonstrate how the here-described workflow can be used for enhancer identification and validation we show step-by-step the selection and characterization of two retinoic acid-dependent enhancers in embryonic stem cells.

<table><tbody><tr><td>KOD DNA polymerase</td><td>Merck Millipore</td><td>71085-3</td><td>for PCR amplification of enhancer from gDNA</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue kit&amp;nbsp;</td><td>Qiagen</td><td>69504</td><td>for genomic DNA isolation</td></tr><tr><td>QIAquick PCR Purification kit</td><td>Qiagen</td><td>28106</td><td>for PCR product purification</td></tr><tr><td>Gel extraction kit&amp;nbsp;</td><td>Qiagen</td><td>28706</td><td>for gel extraction if there are more PCR product</td></tr><tr><td>HindIII</td><td>NEB</td><td>R3104L</td><td>restriction enzyme</td></tr><tr><td>BamHI</td><td>NEB</td><td>R3136L</td><td>restriction enzyme</td></tr><tr><td>FastAP</td><td>Thermo Scientific</td><td>EF0651</td><td>release of 5&#039;- and 3&#039;-phosphate groups from DNA</td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202</td><td>for ligation</td></tr><tr><td>QIAprep Spin Miniprep kit</td><td>Qiagen</td><td>27106</td><td>for plasmid isolation</td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966-021</td><td>ES media</td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070.03</td><td>ES media</td></tr><tr><td>MEM Non-Essential Amino Acid</td><td>Sigma</td><td>M7145</td><td>ES media</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td>ES media</td></tr><tr><td>Beta Mercaptoethanol</td><td>Sigma</td><td>M6250</td><td>ES media</td></tr><tr><td>FuGENE HD&amp;nbsp;</td><td>Promega</td><td>E2311</td><td>transfection reagent</td></tr><tr><td>Opti-MEM&amp;reg; I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-062</td><td>for transfection</td></tr><tr><td>All-trans retinoic acid</td><td>Sigma</td><td>R2625</td><td>ligand, for activation of RAR/RXR</td></tr><tr><td>96-well clear plate</td><td>Greiner</td><td>655101</td><td>for Beta galactosidase assay</td></tr><tr><td>96-well white plate</td><td>Greiner</td><td>655075</td><td>for Luciferase assay</td></tr><tr><td>D-luciferin, potassium salt</td><td>Goldbio.com</td><td>115144-35-9</td><td>for Luciferase assay</td></tr><tr><td>ATP salt</td><td>Sigma</td><td>A7699-1G</td><td>for Luciferase assay</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391-25G</td><td>for Luciferase assay</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-25G</td><td>for Luciferase assay</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>431478-50G</td><td>for Beta galactosidase assay</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;&amp;#8226;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S9638-25G</td><td>for Beta galactosidase assay</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391-25G</td><td>for Beta galactosidase assay</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541-500G</td><td>for Beta galactosidase assay</td></tr><tr><td>ONPG (o-nitrophenyl-&amp;beta;-D-galactosidase)</td><td>Sigma</td><td>N1127-1G</td><td>for Beta galactosidase assay</td></tr><tr><td>TRIzol&amp;reg;</td><td>Life Technologies</td><td>15596-026</td><td>RNA isolation</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Life Technologies</td><td>4368814</td><td>reverse transcription of eRNA</td></tr><tr><td>Rnase-free Dnase</td><td>Promega</td><td>M6101</td><td>Dnase treatment</td></tr><tr><td>SsoFast Eva Green</td><td>BioRad</td><td>750000105</td><td>RT-qPCR mastermix</td></tr><tr><td>CFX384 Touch&amp;trade; Real-Time PCR Detection System</td><td>BioRad</td><td></td><td>qPCR machine</td></tr><tr><td>BioTek Synergy 4 microplate reader</td><td>BioTek</td><td></td><td>luminescent counter</td></tr></tbody></table>

prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

We used a pan-specific RXR antibody in order to identify genome-wide which RA-regulated genes have receptor enrichment in their close proximity. Bioinformatics analysis of RXR ChIP-seq data obtained from ES cells treated with retinoic acid revealed the enrichment of the nuclear receptor half site (AGGTCA) under the RXR occupied sites (Figure 1). Using a bioinformatics algorithm we mapped back the motif search result for the half site to the RXR ChIP-seq data (Figure 2). This analysis helped us to accurately identify those ChIP peaks which were overlapping with canonical nuclear receptor binding sites. Visualization of these sites in IGV indicated enrichment of these transcription factors in the close proximity of Hoxa1, a previously characterized RAR/RXR target (Figure 2 and Figure 3). We also identified a novel RA target gene, namely PRMT8 27. This latter region contains a direct repeat with no spacer nucleotides between the two half sites (AGGTCAAGGTCA)(Figure 3). To functionally validate that the element identified for Hoxa1 can indeed bind RAR/RXR and regulated by RA we constructed TK-luciferase reporter vectors that contained ~300bp genomic region, including the identified elements. We also constructed vectors without the response element (Figure 5). ES cells were transfected and luciferase activity was measured in the absence and presence of retinoic acid (Figure 5). Constructs containing the retinoic acid response element (RARE) did show increased luciferase signal intensity upon RA treatment, while the construct without the RARE was not inducible.
We also studied the retinoic acid-dependent activity of the Hoxa1 enhancer. To ascertain if the sense and anti-sense eRNA expression of the Hoxa1 RAR/RXR-bound enhancer correlate with the mRNA expression of the gene, we also measured the level of Hoxa1 mRNA (Figure 6). These results confirmed that the enhancer activity is induced by short-term RA treatment (3 hr), thus confirming that the enhancer is likely involved in RA-dependent enhancer regulation.
<img alt="Figure 1" src="/files/ftp_upload/53978/53978fig1.jpg" /> 
Figure 1.&#160;Homer De Novo&#160;Motif Results. An output HTML (homerResults.html) generated by Homer for the example RXR ChIP-seq is shown. Sequence logos corresponding to top enriched transcription factor motifs identified by de novo motif discovery at RXR-bound loci. 7463 RXR occupied genomic regions were used for the motif search. 77.66% of these regions contains AGGTCA-like motif (ranked as #1). For detailed description visit:&#160;(http://homer.salk.edu/homer/motif/)&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53978/53978fig2.jpg" /> 
Figure 2. Finding Genomic Regions Containing Motif of Interest. Visualization of the aligned RXR ChIP-seq reads (mm_ES_RXR_24h_ATRA.bam and the AGGTCA motif occurrences (mm_ES_RXR_24h_ATRA_homerpeaks_motif1_mm10s_200_remaped_mbed.bed) by Integrative Genomics Viewer (IGV). Alignments are colored by read strand (reads on + strand: red, - strand: blue). Genome used for the alignment: mm10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53978/53978fig3.jpg" /> 
Figure 3. Genomic Loci of Hoxa1 and PRMT8 Enhancers. IGV snapshot of RXR ChIP-Seq signals obtained from untreated and RA-treated (24 hr, 1 &#181;M) embryonic stem cells 27 are shown. Identified binding sequences are colored red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53978/53978fig4.jpg" /> 
Figure 4. Experimental Design for Testing eRNA Coding Sequences. Enhancers chosen for enhancer RNA (eRNA) measurement should be located at least 1.5 - 2 kb away relative to the transcription start site (TSS) of the closest gene. ChIP-seq data of the transcription factor (TF) of interest and the motif analysis can be used to identify direct binding sites genome-wide. As binding of transcriptional coactivator (e.g., P300) often marks distal enhancers, regions enriched for both the TF and P300 are good enhancer candidates. Expression of eRNAs is positively correlate with the enrichment of activated enhancer histone mark H3K27ac. Regions 200 - 1,000 bp away in sense or anti-sense direction from the center of the transcription factor binding of is recommended for eRNA primer design. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53978/53978fig5.jpg" /> 
Figure 5. Luciferase Activity of the Indicated Reporter Constructs in ES Cells. Indicated genomic regions of the Hoxa1 enhancer were cloned into TK-Luc vector and transfected into embryonic stem cells. Construct 1 and 2 contained the retinoic acid response element (RARE, underlined sequence). Cells were treated with RA (24 hr, 1 &#181;M). &#946;Gal was used as an internal control for normalization. Bars represent mean normalized values from four biological replicates. &#177; s.d. *** P &#60;0.005 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53978/53978fig6.jpg" /> 
Figure 6. Retinoic Acid Induced Enhancer RNA Transcription. Hoxa1 mRNA and eRNA levels as measured by RT-qPCR. Total RNA was isolated from ES cells treated for 3 hr&#160;with retinoic acid (RA) or DMSO as vehicle (veh). Forward primers for the sense and anti-sense eRNA detection and the PCR amplicons are shown. Distance of regions detected by eRNA RT-qPCR from the center of the RXR binding site is indicated. Values were normalized to the average of 36B4 mRNA. Data represent mean &#177; s.e.m. *** P &#60;0.005 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53978/53978fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation at JoVE.com

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

prediction-validation-gene-regulatory-elements-activated-during

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.2K Views.  Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona. In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Video: Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Neuroscience

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

Biology

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

Source: Ababon, M. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54443">Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for assessing retinoic acid activity in mouse embryos. Upon isolating cells from the mouse embryos, the cells are co-cultured with retinoic acid (RA) reporter cells. Embryonic cells produce RA, which induces beta-galactosidase expression in reporter cells, indicating RA activity in the embryonic cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Solution and Media Preparation

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Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

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