This work was supported by Takeda Science Foundation (T.F.); the Asahi Glass Foundation; the Uehara Memorial Foundation (H.F.); the Kurata Memorial Hitachi Science and Technology Foundation (T.F. and H.F.); Grant-in-Aid for Young Scientists (B) (#25830131), Grant-in-Aid for Scientific Research (C) (#15K06895) (T.F.); and a Grant-in-Aid for Scientific Research on Innovative Areas &#39;Transcription Cycle&#39; (#25118512 &#38; #15H01354), Grant-in-Aid for Scientific Research (B) (#15H04329), Grant-in-Aid for Exploratory Research (#26650059) and &#39;Genome Support&#39; (#221S0002) (H.F.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

1. Design of Engineered DNA-binding Molecules Recognizing the Target Locus
<ol>
	<li>For enChIP using CRISPR complexes, use the CRISPRdirect web tool (http://crispr.dbcls.jp) to identify candidate gRNA target sequences in the genomic region of interest described as previously18. This web tool returns 23 bp genomic sites of the form 5&#39;-N20NGG-3&#39; within the target region.</li>
	<li>Synthesize a gBlock including the U6 promoter sequence and the sequence of 5&#39;-N20 in 5&#39;-N20NGG-3&#39; using commercial services (see Figure 2)19,20. For subcloning purposes, include appropriate restriction enzyme sites outside of the gBlock.</li>
	<li>Insert the gBlock into an appropriate vector as previously described16. Several retroviral vectors for gBlocks are available (see Materials).</li>
	<li>For enChIP using TAL proteins, design TAL proteins recognizing the target loci. Generate plasmids encoding TAL proteins using commercial services. Generate expression vectors containing the TAL proteins fused with one or more tags such as 3&#215;FLAG as previously described15.</li>
</ol>
2. Establishment of Cells for the enChIP Analysis
<ol>
	<li>Express 3&#215;FLAG-dCas9 and the gRNA (CRISPR-based procedure) or 3&#215;FLAG-TAL (TAL protein-based procedure) in the cells to be analyzed as described previously14-17.
	<ol>
		<li>Use transient transfection if the transfection efficiency is high. An expression vector containing 3&#215;FLAG-dCas9 and the CMV promoter is available (see Materials).</li>
		<li>Consider establishing stable transformants using conventional methods if the transient transfection efficiency is low. In addition to the aforementioned retroviral vectors for gBlock, retroviral vectors of 3&#215;FLAG-dCas9 are available (see Materials).</li>
	</ol>
	</li>
	<li>Confirm the expression of the 3xFLAG-dCas9 or 3xFLAG-TAL protein in the transfected cells by immunoblot analysis using an anti-FLAG antibody (Ab) as described previously14-17. 
	Note: Expression of these tagged proteins can also be confirmed by intracellular staining with anti-FLAG-FITC and a subsequent FACS analysis. A protocol for intracellular staining can be downloaded from our homepage (http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/iChIP_protocols/english.html).</li>
	<li>Confirm expression of the gRNA by standard RT-PCR technique.</li>
	<li>For quantitative identification of proteins interacting with the genomic region of interest, consider use of a stable isotope labeling by amino acids in cell culture (SILAC) analysis combined with enChIP (enChIP-SILAC). 
	Note: Media for the SILAC analysis can be purchased from commercial vendors. SILAC is powerful in detecting specific interactions.
	<ol>
		<li>Culture cells in SILAC heavy medium. Prepare cells cultured in SILAC light medium as a negative control. Use at least 5 &#215; 107 cells for each SILAC medium (heavy or light). For efficient labeling, add both L-Lysine-2HCl, 13C6 and L-Arginine-HCl, 13C6, 15N4 in the SILAC Heavy media. Note: At least five cell divisions are necessary to label proteins.
		<ol>
			<li>For example, culture human fibrosarcoma HT1080 cells stably expressing 3xFLAG-dCas9 and a gRNA at 37 &#176;C and 5% CO2 in DMEM media for SILAC and dialyzed fetal bovine serum with Lysine-2HCl plus L-Arginine-HCl (Light medium) or 13C6 L-Lysine-2HCl plus 13C615N4 L-Arginine-HCl (Heavy medium) (see Materials)16. Add 50 mg of light or heavy L-Lysine-2HCl and L-Arginine-HCl in 500 ml of medium.</li>
			<li>Trypsinize and replate cells before they become confluent so that cells maintain exponential growth.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Crosslinking of Cells with Formaldehyde
<ol>
	<li>For cells in suspension culture, transfer 2 &#215; 107 cells expressing 3xFLAG-TAL proteins or 3xFLAG-dCas9 plus gRNA and suspend in 30 ml of regular culture medium in a 50 ml centrifuge tube. When more cells are used, increase the volumes and amounts of reagents proportionally. For SILAC experiments, mix the cells cultured in heavy medium or light medium (5 x 107 cells each) and divide the total of 1 x 108 cells into 5 tubes containing 2 x 107 cells.</li>
	<li>Add 810 &#181;l of 37% formaldehyde to 30 ml of the cell suspension (final concentration 1%) and incubate at 37 &#176;C for 5 min. For adherent cells, directly fix cells in culture dishes by adding 810 &#181;l of 37% formaldehyde to 30 ml of culture medium.</li>
	<li>Stop the crosslinking by adding 3.1 ml of 1.25 M glycine solution (final concentration 127 mM), and incubate at RT for 10 min.</li>
	<li>Collect cells by centrifugation (300 &#215; g for 5 min at 4 &#176;C). Carefully discard the supernatant including formaldehyde and store it in an appropriate waste bottle.
	<ol>
		<li>For adherent cells, detach cells with a cell scraper and harvest in a 50 ml tube, and collect cells as described in this step.</li>
		<li>For SILAC experiments, mix the detached cells cultured in heavy medium or light medium (5 x 107 cell each), divide the total of 1 x 108 cells into 5 tubes containing 2 x 107 cells, and collect cells as described in this step.</li>
	</ol>
	</li>
	<li>Wash the cell pellet with 30 ml of PBS twice per tube. Carefully discard the supernatant including formaldehyde and store it in an appropriate waste bottle.Handle the formaldehyde waste according to chemical safety guidelines. The fixed cells can be frozen and stored at -80 &#176;C.</li>
</ol>
4. Preparation of Chromatin (per 2 x 107 cells)
<ol>
	<li>Suspend the fixed cells in 10 ml of cell lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5% IGEPAL CA-630, and 1&#215; protease inhibitors) and incubate on ice for 10 min.</li>
	<li>Centrifuge the sample (830 &#215; g at 4 &#176;C for 8 min) and discard the supernatant carefully.</li>
	<li>Suspend the pellet in 10 ml of nuclear lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.5% lauroylsarcosine, and 1x protease inhibitors). Incubate on ice for 10 min and vortex every 2-3 min.</li>
	<li>Centrifuge the sample (830 &#215; g at 4 &#176;C for 8 min) and discard the supernatant carefully.</li>
	<li>Wash the pellet in 10 ml of PBS. The pellet (chromatin fraction) can be stored at -80 &#176;C after immediate freezing in liquid nitrogen.</li>
</ol>
5. Sonication of Chromatin (per 2 x 107 cells)
<ol>
	<li>Suspend the chromatin fraction in 800 &#181;l of modified lysis buffer 3 (10 mM Tris (pH 8.0), 1 mM EDTA, 150 mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, and 1x protease inhibitors) and transfer into a 1.5 ml tube.</li>
	<li>Sonicate the chromatin using a sonicator (see Materials) and the following conditions: output: 3; duty: 100% (continuous); and time: free. Perform 10-18 cycles of sonication for 10 sec and cooling on ice for 20 sec. To avoid excessive heating, incubate the samples on ice for 2 min every 5-6 cycles. To avoid foaming, keep the position of the tip of the sonication probe 0.5 cm above the bottom of the tube.</li>
	<li>Centrifuge the sample at (16,000 &#215; g at 4 &#176;C for 10 min) and transfer the supernatant into a 1.5 ml tube. The sonicated chromatin can be stored at -80 &#176;C after immediate freezing in liquid nitrogen.</li>
</ol>
6. Evaluation of Chromatin Fragmentation
<ol>
	<li>Mix 10 &#181;l of the fragmented chromatin with 85 &#181;l of distilled water.</li>
	<li>Add 4 &#181;l of 5 M NaCl and incubate at 65 &#176;C O/N.</li>
	<li>Add 1 &#181;l of 10 mg/ml RNase A and incubate at 37 &#176;C for 45 min.</li>
	<li>Add 2 &#181;l of 0.5 M EDTA (pH 8.0), 4 &#181;l of 1 M Tris (pH 6.8), and 1 &#181;l of 20 mg/ml Proteinase K, and then incubate at 45 &#176;C for 1.5 hr.</li>
	<li>Separate 10 &#181;l of the sample by electrophoresis in a 1% agarose gel that does not contain staining dyes such as SYBR Green.</li>
	<li>Stain the gel to evaluate the distribution of the lengths of the fragmented chromatin. Conditions that generate an average length of 0.5-2 kbp (range of 0.2-4 kbp) are recommended.</li>
	<li>Purify DNA from the remaining samples by phenol-chloroform extraction or by using a DNA extraction kit (see Materials). The purified DNA can be used as input DNA to estimate the yields of enChIP analyses (see 8.11).</li>
</ol>
7. Preparation of Dynabeads Conjugated with Antibodies (per 2 x 107 cells)
<ol>
	<li>Prepare two 1.5 ml tubes, one for pre-clearing using normal mouse IgG and the other for incubation with the anti-FLAG Ab. Add 150 &#181;l of Protein G-conjugated magnetic beads (see Materials) to each tube.</li>
	<li>Place the tubes on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.</li>
	<li>Suspend the beads in 1 ml of PBS containing 0.01% Tween-20 (PBS-T). Place the tubes on a magnetic stand and wait for 2 min. Discard the supernatant by pipetting, and then repeat the step.</li>
	<li>Suspend the beads in 1 ml of PBS-T containing 0.1% BSA.</li>
	<li>Add 15 &#181;g of normal mouse IgG or anti-FLAG Ab and rotate at 4 &#176;C O/N.</li>
	<li>Spin down briefly, then place the tubes on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.</li>
	<li>Suspend the beads in 1 ml of PBS-T. Invert the sample several times and spin down briefly. Place the tubes on a magnet stand and wait for 3 min, then discard the supernatant by pipetting. Repeat the wash two more times with PBS-T (total of three wash steps).</li>
</ol>
8. Chromatin Immunoprecipitation (per 2 x 107 cells)
<ol>
	<li>Mix the fragmented chromatin prepared in 5.3) (approximately 800 &#181;l) with one-fifth of the volume (approximately 200 &#181;l) of modified lysis buffer 3 containing 5% Triton X-100 (final concentration of 1% Triton X-100 ).</li>
	<li>Add the chromatin solution to the tube in which Protein G-conjugated magnetic beads conjugated with normal mouse IgG were prepared. Rotate at 4 &#176;C for 1 hr.</li>
	<li>Place the tube on a magnet stand and wait for 3 min.</li>
	<li>Transfer the supernatant into the tube in which Protein G-conjugated magnetic beads conjugated with anti-FLAG Ab were prepared. Rotate at 4 &#176;C O/N.</li>
	<li>Place the tube on a magnet stand and wait for 3 min. Discard the supernatant by pipetting.</li>
	<li>Suspend the beads in 1 ml of low salt buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1x protease inhibitors) and rotate at 4 &#176;C for 5 min. Place the tube on a magnet stand and wait for 3 min. Discard the supernatant by pipetting and repeat the wash step.</li>
	<li>Wash the beads with high salt buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1x protease inhibitors) twice.</li>
	<li>Wash the beads with LiCl buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, and 1x protease inhibitors) twice.</li>
	<li>Wash the beads with TBS-IGEPAL CA-630 (50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% IGEPAL CA-630, and 1x protease inhibitors) once.</li>
	<li>Suspend the beads in 200 &#181;l of elution buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% IGEPAL CA-630, 1x protease inhibitors, and 500 &#181;g/ml 3&#215;FLAG peptide) and incubate at 37 &#176;C for 20 min. Place the tube on a magnet stand and wait for 3 min. Transfer the supernatant into a new 1.5 ml tube and repeat the elution step.</li>
	<li>Purify DNA from small portion (e.g.,&#160;5%) of the eluate by phenol-chloroform extraction or by using a DNA extraction kit (see Materials). The purified DNA can be used for PCR with specific primer sets to estimate the yields of enChIP analyses by comparing with input DNA prepared in Step 6.7 as described previously14,16.</li>
</ol>
9. SDS-PAGE, Staining, and Mass Spectrometric Analysis
<ol>
	<li>Mix the eluate (400 &#181;l) with 1 ml of 2-propanol, 50 &#181;l of 3M sodium acetate (pH 5.2), and 5 &#181;l of 20 mg/ml glycogen. Precipitate the chromatin at -20 &#176;C O/N.</li>
	<li>Centrifuge the sample (16,000 &#215; g at 4 &#176;C for 30 min) and discard the supernatant. Rinse the pellet with 1 ml of 70% ethanol and centrifuge again (16,000 &#215; g at 4 &#176;C for 10 min). Discard the supernatant completely by pipetting.</li>
	<li>Suspend the pellet in 40 &#181;l of 2x sample buffer (125 mM Tris (pH 6.8), 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, and 0.004% bromophenol blue). Vortex for 5 min to dissolve the pellet completely, and then incubate at 100 &#176;C for 30 min to denature the proteins and reverse the crosslinking.</li>
	<li>For SDS-PAGE, run 40 &#181;l of the sample on the gel until the dye reaches 1 cm below the well. Note: We usually use 4-20% gradient gels, but other % gels can be used.</li>
	<li>Stain the gel with Coomassie Brilliant Blue or silver stain.</li>
	<li>Cut the gel into five pieces (2 mm height).</li>
	<li>Perform in gel digestion and mass spectrometric analysis as described previously13-16.</li>
	<li>For SILAC experiments with 5 x 107 cells each (total of 1 x 108 cells), suspend pellet from the initial five tubes in 40 &#181;l of 2x sample buffer (it is not necessary to scale up).</li>
</ol>
10. Purification of RNA and RNA-Seq Analysis
<ol>
	<li>To purify the RNA after enChIP, add 5 U/ml of RNase Inhibitor (see Materials) to all of the buffer solutions, except modified lysis buffer 3 and the elution buffer, to which add 40 U/ml of RNase Inhibitor.</li>
	<li>Mix the eluate (400 &#181;l) with 16 &#181;l of 5 M NaCl and incubate at 65 &#176;C for 2 hr.</li>
	<li>Add 1 ml of acid-guanidinium-phenol-based reagent (see Materials) to the sample. Vortex for 15 sec and then incubate at RT for 5-15 min. Centrifuge the sample&#160;for 15 min at 12,000 &#215; g and RT.</li>
	<li>Transfer the supernatant to a new 1.5 ml tube and add 5 &#181;l of p-bromoanisole. Vortex for 15 sec and then incubate at RT for 3-5 min. Centrifuge the sample for 10 min at 12,000 &#215; g and RT.</li>
	<li>Transfer the supernatant (approximately 1 ml) into a new 2 ml tube and add 1 ml of 2-propanol. Invert the tube and incubate at RT for 10 min. Load the mixture onto a column in an RNA purification kit (see Materials). Centrifuge the column for 1 min at 12,000 &#215; g and RT.</li>
	<li>Wash the column with 400 &#181;l of RNA Wash Buffer in an RNA purification kit (see Materials).</li>
	<li>Add 80 &#181;l of DNase I cocktail (mixture of 5 &#181;l of DNase I (1 U/&#181;l), 8 &#181;l of 10&#215; DNase I Reaction Buffer, 3 &#181;l of DNase/RNase-free water, and 64 &#181;l of RNA Wash Buffer in an RNA purification kit (see Materials)) to the column. Incubate the sample at 37 &#176;C for 15 min and then centrifuge &#160;for 30 sec at 12,000 &#215; g and RT.</li>
	<li>Wash the column with 400 &#181;l of RNA PreWash Buffer in an RNA purification kit (see Materials) twice.</li>
	<li>Elute the RNA with 50 &#181;l of DNase/RNase-free water. The eluted RNA can be used for RNA sequencing.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+analysis+of+Cas9+binding+specificity+using+ChIP-seq+and+targeted+sequence+capture.">A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture.</a> Nucleic Acids Res. 43, 3389-3404 (2015).</li><li>de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shelterin:+the+protein+complex+that+shapes+and+safeguards+human+telomeres.">Shelterin: the protein complex that shapes and safeguards human telomeres.</a> Genes Dev. 19, 2100-2110 (2005).</li></ol>

The authors have filed a patent on enChIP (Patent name: 'Method for isolating specific genomic regions using DNA-binding molecules recognizing endogenous DNA sequences'; Patent number: PCT/JP2013/74107). H.F. is a member of the Advisory Board of Addgene.

Here, we describe the purification of specific genomic regions using engineered DNA-binding molecules such as the CRISPR system and TAL proteins, and the identification of proteins and RNAs bound to these genomic regions. Binding of engineered DNA-binding molecules to the genome may affect chromatin structure, including nucleosome positioning, and may abrogate genomic functions, as described in CRISPR interference experiments21. To avoid these potential aberrant effects, we propose specific guidelines for choosing target genomic regions. First, to avoid potential inhibition of the recruitment of RNA polymerases and transcription factors, as well as disruption of nucleosome positioning around the transcription start site, the target regions for analyses of promoter regions should be several hundred base pairs upstream of (5' to) the transcription start site. By contrast, when analyzing genomic regions with distinct boundaries, such as enhancers and silencers, genomic regions that are directly juxtaposed to these regions can be targeted because it is less likely that the binding of engineered DNA-binding molecules will affect their functions. Furthermore, it is best to avoid using target regions that are conserved among different species, because important DNA-binding molecules often bind to evolutionarily conserved regions and inhibition of their binding might disrupt the functions of the target genomic regions. In this regard, it is always necessary to check that the function of the target genomic region is maintained in the established cells used for enChIP analyses. Because multiple gRNAs can be tested easily and it is tedious and expensive to generate multiple versions of TAL or zinc-finger proteins recognizing different target genomic regions, enChIP using CRISPR is more advantageous than enChIP using other proteins.
It has been shown that dCas9 binds to off-target sites although affinity to those sites might be weaker than that to the target sites22-25. There are several ways to manage contamination of molecules bound to those off-target sites. First, the use of several, at least two, different gRNAs would be recommended. Those molecules commonly observed in enChIP using distinct gRNAs would be true positives. Second, comparison of different conditions for enChIP would be effective in cancelling contamination of non-specific molecules and molecules bound to off-target sites. Examples of those comparison sets would be (i) stimulation (-) and (+), or (ii) different cell types such as T cells vs. B cells. Finally, quantitative analysis of binding of candidate molecules should be performed to confirm their specific binding to the target sites. It is preferable to prepare cells expressing only dCas9 but not gRNA as a negative control.
Using enChIP analyses, we were able to successfully identify a number of known and novel molecules interacting with specific genomic regions (Tables 1-3)14-17. However, this technique failed to detect some other known proteins interacting with these regions. For example, STAT1 reportedly associates with the IRF-1 promoter upon IFN&#947; stimulation8, but our enChIP-SILAC analysis did not detect STAT1 as a protein induced to interact with this genomic region16. In addition, in the enChIP-MS analysis of telomeres, we did not detect shelterin proteins consisting of TRF-1 and TRF-215, which have been shown to interact with telomeres26. There are a few potential reasons for these discrepancies. First, the stoichiometry of binding of Stat1 to the IRF-1 promoter might be very low. It is reasonable that enChIP-MS, including enChIP-SILAC, detects proteins that are more abundantly associated with target genomic regions; hence, the analysis of more cells might be necessary to detect these proteins. Increases in the sensitivities of MS instruments would also contribute to the efficient detection of proteins with low stoichiometric binding. Second, some proteins, possibly including Stat1 and shelterins, might be difficult targets for MS analyses. Third, in our analysis of telomere-binding proteins15, the 3&#215;FLAG-TAL proteins recognizing telomeres (3&#215;FN-Tel-TAL) might have blocked the binding of shelterins to telomeres in a competitive fashion.
In contrast to the relative difficulty of detecting transcription factors binding to specific genomic regions using enChIP, we successfully identified epigenetic regulators such as histone modification enzymes using enChIP analyses. The success of this technique may be due to the fact that epigenetic regulators bind to a broad range of genomic regions; hence, more proteins per genomic region are available for MS. Because epigenetic regulators are increasingly recognized as important targets for drugs against intractable diseases such as cancer, enChIP would be a useful tool for the identification of epigenetic drug targets.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of genomic functions such as transcription and epigenetic regulation. Although several techniques have been developed for biochemical analysis of specific genomic regions1-7, they are not used widely at this stage because of their intrinsic problems such as limited application (e.g., only for high-copy number loci or loci with repeats) and too much time and efforts required.
In order to perform biochemical analysis of specific genomic regions easily, we have developed two locus-specific chromatin immunoprecipitation (ChIP) technologies, namely insertional ChIP (iChIP)8-13 and engineered DNA-binding molecule-mediated ChIP (enChIP)14-17. In iChIP, a locus of interest is tagged by inserting recognition sequences of an exogenous DNA-binding protein such as LexA. The locus is then isolated by affinity purification using the tagged DNA-binding protein. In enChIP, engineered DNA-binding molecules, such as zinc-finger proteins, transcription activator-like (TAL) proteins, and clustered regularly interspaced short palindromic repeats (CRISPR) complexes, are used to tag a locus of interest (Figure 1). Subsequently, the genomic region is isolated by affinity purification of the tagged DNA-binding molecules.
One of the advantages of enChIP over iChIP is that insertion of recognition sequences of an exogenous DNA-binding protein is not necessary. Targeting of loci using CRISPR complexes consisting of a catalytically inactive form of Cas9 (dCas9) and a guide RNA (gRNA) is much easier than targeting of these regions by iChIP or enChIP using TAL and zinc-finger proteins. Here, we describe a step-by-step protocol for enChIP combined with mass spectrometry and RNA sequencing (RNA-Seq) to identify locus-associated proteins and RNAs, respectively.

<table border="0" cellpadding="0" cellspacing="0" width="964">
	<tbody>
		<tr height="38">
			<td height="38" style="height:38px;width:239px;">gBlock synthesis service</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">Gene Synthesis by GeneArt</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:239px;">gBlock synthesis service</td>
			<td style="width:199px;">IDT (Integrated DNA Technologies)</td>
			<td style="width:175px;">gBlocks Gene Fragments</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-neo</td>
			<td>Addgene</td>
			<td style="width:175px;">51128</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-GFP</td>
			<td>Addgene</td>
			<td style="width:175px;">51134</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-DsRed-Express2</td>
			<td>Addgene</td>
			<td style="width:175px;">51135</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-hCD2</td>
			<td>Addgene</td>
			<td style="width:175px;">51143</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">TAL synthesis service</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">GeneArt Precision TALs</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">3xFLAG-dCas9/pCMV-7.1</td>
			<td>Addgene</td>
			<td style="width:175px;">47948</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-puro</td>
			<td>Addgene</td>
			<td style="width:175px;">51240</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-IG</td>
			<td>Addgene</td>
			<td style="width:175px;">51258</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-I2</td>
			<td>Addgene</td>
			<td style="width:175px;">51259</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-neo</td>
			<td>Addgene</td>
			<td style="width:175px;">51260</td>
			<td style="width:353px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">anti-FLAG M2 Ab</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:175px;">F1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">FITC-conjugated anti-FLAG M2</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:175px;">F4049</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">DMEM medium for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89985</td>
			<td>Other medium can be purchased from Life Technologies</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">Dialyzed FBS for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89986</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">L-Lysine-2HCl for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89987</td>
			<td>for Light medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">L-Arginine-HCl for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89989</td>
			<td>for Light medium</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:239px;">L-Lysine-2HCl, 13C6 for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89988</td>
			<td>For Heavy medium</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:239px;">L-Arginine-HCl, 13C6, 15N4 for SILAC</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89990</td>
			<td>For Heavy medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Complete, mini, EDTA-free</td>
			<td style="width:199px;">Roche Diagnostics</td>
			<td style="width:175px;">4693159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ultrasonic Disruptor UD-201</td>
			<td style="width:199px;">Tomy Seiko</td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChIP DNA Clean &#38; Concentrator</td>
			<td style="width:199px;">Zymo Research</td>
			<td style="width:175px;">D5205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Dynabeads-Protein G</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">DB10004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">RNasin Plus RNase Inhibitor</td>
			<td style="width:199px;">Promega</td>
			<td style="width:175px;">N2611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Isogen II</td>
			<td style="width:199px;">Nippon Gene</td>
			<td style="width:175px;">311-07361</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Direct-zol RNA Miniprep kit</td>
			<td style="width:199px;">Zymo Research</td>
			<td style="width:175px;">R2050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">LTQ Orbitrap Velos</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">nanoLC</td>
			<td style="width:199px;">Advance, Michrom Bioresources</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">HTC-PAL autosampler</td>
			<td style="width:199px;">CTC Analytics</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
		</tr>
	</tbody>
</table>

isolation of specific genomic regions and identification of associated molecules by enchip

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

Overall, 1-30% of target genomic regions can be purified using enChIP. Figure 3 includes representative data showing the yields of enChIP analyses targeting telomeres and the promoter of the interferon (IFN) regulatory factor-1 (IRF-1) gene. As examples of typical results, Table 1 lists the proteins associated with the IRF-1 promoter in an IFN&#947;-specific manner identified by enChIP-SILAC, Table 2 lists the telomere-binding proteins identified by enChIP combined with mass spectrometry (enChIP-MS), and Table 3 lists the RNAs associated with telomeres identified by enChIP-RNA-Seq.
<img alt="Figure 1" src="/files/ftp_upload/53478/53478fig1.jpg" /> 
Figure 1. Overview of enChIP. enChIP using CRISPR (A, B) and TAL (C, D, E) is shown. A locus of interest is tagged with engineered DNA-binding molecules such as a TAL protein or the CRISPR system consisting of a catalytically inactive form of Cas9 (dCas) and guide RNA (gRNA). The molecular interactions are fixed with formaldehyde or other crosslinkers, if necessary. Subsequently, fixed chromatin is fragmented by sonication or enzymatic digestion. The tagged genomic regions are purified by affinity purification. Finally, crosslinking is reversed, and interacting molecules (genomic regions, RNAs, and proteins) are identified using next generation sequencing and mass spectrometry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53478/53478fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53478/53478fig2.jpg" /> 
Figure 2. Sequence of gBlock. The U6 promoter (in black), the G nucleotide before the guide sequence (in blue), the guide sequence (gRNA spacer) (in red), the scaffold sequence (in green), and the terminator sequence (in orange) are shown.
<img alt="Figure 3" src="/files/ftp_upload/53478/53478fig3.jpg" /> 
Figure 3. Yields of representative enChIP analyses. (A) Percentage inputs for the target IRF-1 locus and the non-target Sox2 locus. (B) Percentage inputs for the target telomeres and non-target &#947;-satellites. The figures have been adapted and modified from previous publications15,16.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Categories</td>
			<td>Proteins</td>
		</tr>
		<tr>
			<td>Transcription</td>
			<td>DDX1, PARP1, CKAP4, Pescadillo homolog, PUR&#946;, 
			activated RNA polymerase II transcriptional activator p15, 
			BTF3, Myb-binding protein 1A</td>
		</tr>
		<tr>
			<td>Histone&#160; deacetylation, 
			corepressor components</td>
			<td>RBBP4, PA2G4, TBL3</td>
		</tr>
		<tr>
			<td>Acetyltransferase</td>
			<td>Protein arginine N-methyltransferase 1</td>
		</tr>
		<tr>
			<td>DNA topoisomerase</td>
			<td>DNA topoisomerase 2&#945;</td>
		</tr>
		<tr>
			<td>Histones</td>
			<td>Histone H2A.Z, histone H3.2</td>
		</tr>
	</tbody>
</table>
Table 1: Examples of proteins associated with the human IRF-1 promoter region in an IFN&#947;-specific manner identified by enChIP-SILAC. The table has been adapted from a previous publication16.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Categories</td>
			<td>Proteins</td>
		</tr>
		<tr>
			<td>Mammalian telomere-binding proteins</td>
			<td>PML, RPA, CDK1, PARP1, PCBP1</td>
		</tr>
		<tr>
			<td>Telomere-binding proteins in yeast 
			or other organisms</td>
			<td>IMP4</td>
		</tr>
		<tr>
			<td>Proteins interacting with telomere-binding 
			proteins [associated telomere-binding protein]</td>
			<td>DNA polymerase &#945; (POLA1) [Cdc13p], 
			ARMC6 [TRF2], CTBP1 [FoxP2-POT1], 
			exportin-5 [TERT], GNL3L [TRF1], 
			exportin-1 [TERT], 14-3-3 [TERT]</td>
		</tr>
		<tr>
			<td>Proteins localizing to heterochromatin</td>
			<td>BEND3</td>
		</tr>
		<tr>
			<td>Proteins regulating epigenetic marks</td>
			<td>KDM5C</td>
		</tr>
		<tr>
			<td>Proteins whose mutations affect telomere function</td>
			<td>DNA polymerase &#945; (POLA1), HAT1, Nup133, 
			CDK7, DPOE1, PRDX1, TYSY, 
			glutamate-cysteine ligase, glutaredoxin, SMRC1</td>
		</tr>
	</tbody>
</table>
Table 2: Examples of proteins associated with mouse telomeres identified by enChIP-MS. The table has been adapted from a previous publication15.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Categories</td>
			<td>RNAs</td>
		</tr>
		<tr>
			<td>Telomerase components</td>
			<td>Terc, Rmrp</td>
		</tr>
		<tr>
			<td>Telomeric RNAs</td>
			<td>TERRAs</td>
		</tr>
		<tr>
			<td>scaRNAs</td>
			<td>Scarna6, Scarna10, Scarna13, Scarna2</td>
		</tr>
		<tr>
			<td>H/ACA snoRNAs</td>
			<td>Snora23, Snora74a, Snora73b, Snora73a</td>
		</tr>
		<tr>
			<td>C/D snoRNAs</td>
			<td>Snord17, Snord15a, Snord118</td>
		</tr>
		<tr>
			<td>lncRNA</td>
			<td>Neat1</td>
		</tr>
	</tbody>
</table>
Table 3: Examples of RNAs associated with mouse telomeres identified by enChIP-RNA-Seq. The table has been adapted from a previous publication17.

Watch this Scientific Journal Video about Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP at JoVE.com

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

isolation-specific-genomic-regions-identification-associated

Nanyang Technological University

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JoVE Journal

Biology

10.4K Views.  Osaka University. The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Video: Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

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By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...