The authors wish to thank the members of the Harvey Cushing Neuro Oncology Laboratory for support and constructive criticism. This work was supported by NINDS grants K12NS80223 and K08NS101091 to P. P.

1. Characterization of Functionally Associated miRNAs in Glioblastoma
<ol>
	<li>Analysis of broad differential miRNA expression in glioblastoma vs. brain
	<ol>
		<li>First, determine the most significantly deregulated miRNAs in the tumor. This can be achieved using at least three different methods:
		<ol>
			<li>Mine the Cancer Genome Atlas, found at https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga, for sequencing data11.</li>
			<li>Perform microarray analysis from a fresh operative specimen12.</li>
			<li>Use previously published datasets13. 
			NOTE: Whichever method is chosen, the output provides a bulk set of miRNAs whose expression is statistically correlated (either directly or inversely) with tumor biology. This initial group constitutes the pool of miRNAs that are further analyzed for the identification of a subset of miRNAs displaying the most stringent functional association by performing a more dynamic analysis of expression changes, as discussed below.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Condition-specific miRNA expression analysis
	<ol>
		<li>Induction of cell differentiation:
		<ol>
			<li>Use pre-coated poly-D-lysine (PDL) plastic dishes to favor the formation of a monolayer culture. For dish coating, dissolve PDL in water at a concentration of 100 &#181;g/mL. Use 1 mL of solution per 25 cm2 surface. Rinse 2x with PBS 6 h after application of the PDL solution.</li>
			<li>Plate human neural stem cells in equal amounts (100,000 cells/5 mL) either in stem cell medium (neurobasal medium + B27 supplement + 20 ng/mL EGF/FGF), astrocytic differentiation medium (DMEM + 10% FBS), or neuronal differentiation medium (neurobasal medium + B27 supplement, + 2 &#181;M retinoic acid)14. The protocol for oligodendrocyte differentiation requires addition of IGF-1 (200 ng/mL) after EGF/FGF removal14,15.</li>
			<li>After cell plating, place in incubator for 1 week at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>RNA extraction:
		<ol>
			<li>After 7 days, remove the cells from the incubator and remove the medium.</li>
			<li>Wash the cells with PBS (5 mL). Then, add 1 mL of lysis reagent (see Table of Materials) to lyse the cells and scrape the cells into 1.5 mL microfuge tube using a cell scraper. Keep the tubes containing the lysed cells at room temperature (RT) for 5 min.</li>
			<li>Proceed to total RNA isolation following the manufacturer&#39;s instructions.</li>
		</ol>
		</li>
		<li>miRNA expression analysis:
		<ol>
			<li>Quantify the differential expression of specific miRNAs identified in step 1.1 among the three differentiation patterns by real-time PCR, using TaqMan probes and following the manufacturer&#39;s protocols for reverse transcription and PCR amplification (Figure 1).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Validation of miRNA clusters by stress-specific challenges
	<ol>
		<li>Culture G34 glioblastoma stem-like cells in neurobasal medium + B27 supplement + 20 ng/mL EGF/FGF. Start with 1 x 106 cells in 5 mL in a 25 cm2 low attachment flask.</li>
		<li>Starting 48 h after plating, add doubling concentrations of DNA alkylating agent temozolomide (TMZ) every 5 days, as follows:
		<ol>
			<li>Start with 5 &#181;M for 5 days. After 5 days, remove the medium and replace with fresh medium without temozolomide, to allow the surviving cells to recover. After 48 h, add 10 &#181;M TMZ and incubate for another 5 days.</li>
			<li>Repeat step 1.3.2.1, doubling TMZ concentration each time, until a concentration of at least 100 &#181;M is reached and cells become resistant to the drug10.</li>
		</ol>
		</li>
		<li>In a parallel experiment, irradiate G34 cells as follows:
		<ol>
			<li>Starting 48 h after plating 1 x 106 cells in 5 mL in a 25 cm2 low attachment flask, irradiate cells with 2 Gy of energy (any irradiator providing photon emission is acceptable). Return the cells to the incubator and do not disturb. After 48 h, irradiate the cells again with additional 2 Gy of energy.</li>
			<li>Repeat step 1.3.3.1 4x until a total of 10 Gy of energy is administered.</li>
			<li>Return the cells to the incubator and let them recover in fresh medium for at least 5 days before downstream analysis.</li>
		</ol>
		</li>
		<li>After the induction of resistance, lyse cells using lysis reagent and extract total RNA according to the manufacturer&#39;s protocol.</li>
		<li>Analyze the expression of the specific miRNAs identified in sections 1.1-1.2 as described in step 1.2.1 (Figure 2).</li>
	</ol>
	</li>
	<li>Characterization of the functional convergence of clustered miRNAs
	<ol>
		<li>Obtain the predicted targetome of each miRNAs defined in sections 1.2-1.3 obtained using miRNA targeting prediction tools. 
		NOTE: We generally resort to TargetScan, as its algorithm is periodically updated16. Additional prediction tools are miRanda17, miRDB18, and DIANA-miRPath19. All these programs are free, web-based applications.
		<ol>
			<li>From the front page of Targetscan, select the miRNA of interest from the pre-populated drop-down menu. Click the Submit button.</li>
			<li>Download the resulting list of targets as a spreadsheet using the Download table link (Supplementary Figure 1).</li>
			<li>To decrease the chances of false positive predictions, include only targets within the conserved sites column in downstream analyses. Also, further stringency can be obtained by using additional miRNA prediction programs (see above) and only including targets that are common to all algorithms.</li>
		</ol>
		</li>
		<li>Classify the resulting targetomes according to Gene Ontology categories using ToppGene Suite20 to evaluate for the enrichment of pathways that are common to each miRNA.
		<ol>
			<li>Paste the list of targets obtained from step 1.4.1 in the &#34;Training gene set&#34; window, using HGNC symbol as the entry type. Click Submit &#62; Start. The program will provide an output table showing the most significant &#34;Go&#34; categories for the entered list of genes (Supplementary Figure 2).</li>
		</ol>
		</li>
		<li>To finally establish the contribution of each miRNA to the regulation of a common pathway or cellular process (in this case, chromatin regulation), check each targetome obtained in step 1.4.1 against the full list of genes involved in the specific cellular process (i.e., chromatin regulation), using the Venn diagram function provided in the Bioinformatics and Evolutionary Genomics website http://bioinformatics.psb.ugent.be/webtools/Venn. 
		NOTE: This program allows the intersection of multiple groups at the same time (Figure 3). The specific unique targets provided by each miRNA are then annotated and selected for downstream functional experiments.
		<ol>
			<li>In the front page of the program, upload or copy/paste the list if target mRNAs for each miRNA or interest in the respective windows. Name each list with a unique identifier.</li>
			<li>Click Submit. The program will provide a visual output of a Venn diagram with numbers of genes in each sector, as well as a full list of mRNAs for each subset and intersection combinations.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Assembly of miRNA Modules into a Polycistronic Transgenic Cluster
<ol>
	<li>Preparation of transgenic scaffold based on miR-17-92 cluster locus
	<ol>
		<li>Obtain the nucleotide sequence of the miR-17-92 cluster from the Ensembl genome browser21. Select the ~800 base pair &#34;core&#34; sequence encompassing all six encoded miRNA hairpins of the locus and at least 200-nucleotide flanking sequences both upstream (5&#39;) and downstream (3&#39;) of the core sequence.</li>
		<li>Paste the sequence above into any word editing program.</li>
		<li>Define the sequence of each one of the six native hairpins by retrieving them in miRBase22. Mark each one of these sequences within the span of the previously identified &#34;core&#34; sequence. Any sequences between each hairpin represent spacer sequences, which are important for the correct processing of the transgene.</li>
		<li>Remove the native hairpin sequences from the &#34;core&#34; sequence, with the exception of 3-5 nucleotides at both the 5&#39; and 3&#39; ends of each hairpin, which will serve as an acceptor for the new hairpins.</li>
	</ol>
	</li>
	<li>Building a new transgene encoding multiple miRNAs of choice
	<ol>
		<li>Obtain hairpin sequences of miRNAs intended to be overexpressed in the transgene from miRBase. Carefully note the specific nucleotides that are the sites of microprocessor cleavage.</li>
		<li>Replace the removed hairpins (step 2.1.4) with the hairpin sequences of the desired miRNAs obtained in step 2.2.1.</li>
		<li>Add desired restriction sequences at both flanking regions of the transgene to facilitate subcloning into delivery vectors of choice. Verify that the chosen restriction sequences are not present within the sequence itself.</li>
		<li>For negative controls, use 20-nucleotide scrambled sequences to replace the natural 20-nucleotide sequence of each mature miRNA. Generate these scrambled sequences using an online tool such as https://genscript.com/tool/create-scrambled-sequence.</li>
	</ol>
	</li>
	<li>Verifying the two-dimensional structure of the transgene
	<ol>
		<li>Copy the full transgenic sequence into the RNA structure prediction software program RNAweb Fold23.</li>
		<li>Using standard program settings, click Proceed.</li>
		<li>Analyze the graphical output, particularly for the presence of well-defined hairpins and presence of double-stranded stem structures at least 11 nucleotides proximal to the microprocessor cleavage site. Also, look for the absence of branching points within the hairpin sequences (Figure 4).</li>
	</ol>
	</li>
</ol>
3. Obtaining Transgenes by DNA Synthesis
<ol>
	<li>After the design and in silico validation of the chimeric miRNA cluster, obtain the working sequence using DNA synthesis from commercial vendors24 and proceed with downstream cloning into vectors and transgene delivery. We have used lentiviral vectors for in vitro delivery10 and adeno-associated viruses (AAVs) for in vivo intracranial delivery25.</li>
</ol>

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The authors report no conflicts of interest.

This protocol is based on the notion that rather than functioning in isolation, miRNAs are biologically relevant by working in groups, and these groups are transcriptionally determined by specific cellular contexts26. To justify this approach from a translational perspective, a follow-up protocol that allows recreation of this multi-miRNA pattern in cells/tissues is introduced. This is possible by taking advantage of the relatively simple biogenesis of miRNAs, whereby the recognition of the charac...

miRNAs offer an unmatched opportunity for the development of a broad gene therapy approach to many diseases1,2,3, including cancer4,5. This is based on several unique features of these biological molecules, including their small size6, simple biogenesis7, and natural tendency to function in association8. Many diseases are characterized by specific miRNA expression patterns, which often converge on the regulation of complex biological functions9. The purpose of this method is first to define a strategy to identify groups of miRNAs that are synergistically relevant for specific cellular functions. Consequently, it provides a strategy for the re-establishment of such miRNA combinations in downstream studies and applications.
This method allows for functional analysis of multiple miRNAs at once, leveraging on their simultaneous targeting of a large number of mRNAs, thus recapitulating the complex landscapes of diseases. This approach has been recently employed to define a group of three miRNAs that 1) are simultaneously downregulated in brain cancer and 2) show a strong co-expression pattern during neural differentiation as well as in response to genotoxic stress by radiation or a DNA alkylating agent. The combinatorial re-expression of this module of three miRNAs by the clustering method described below results in profound interference with the biology of cancer cells and can be easily used as a gene therapy strategy for preclinical studies10. This protocol may be of particular interest to those involved in miRNA research and its translational applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4% low melting temperature agarose&#160;</td>
			<td>IBI Scientific</td>
			<td>IB70058</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;M sterile filter unit</td>
			<td>Merck Millipore</td>
			<td>SLH033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5-mL Microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well plates&#160;</td>
			<td>Greiner Bio-One</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Athymic mice (FoxN1 nu/nu)</td>
			<td>Envigo</td>
			<td>069(nu)/070(nu/+)</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>Greiner Bio-One</td>
			<td>660175</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper, 16cm</td>
			<td>Sarstedt</td>
			<td>83.1832</td>
			<td></td>
		</tr>
		<tr>
			<td>Cesium 137 irradiator&#160;</td>
			<td>JL Sheperd and Associates</td>
			<td>Core Facility (Harvard Medical School)</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>439142-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11995040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline&#160;</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E4009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-293</td>
			<td>American Type Culture Collecti</td>
			<td>ATCC CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin solution</td>
			<td>Sigma-Aldrich</td>
			<td>1051750500</td>
			<td></td>
		</tr>
		<tr>
			<td>Human primary glioma stem-like cells (GBM62)</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. E. A. Chiocca (Brigham and Women&#8217;s Hospital, Boston, MA)</td>
		</tr>
		<tr>
			<td>Human primary glioma stem-like cells (MGG4)</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. Hiroaki Wakimoto (Massachusetts General Hospital, Boston, MA)</td>
		</tr>
		<tr>
			<td>Lentiviral vector pCDH-CMV-MCS-EF1-copGFP</td>
			<td>System Biosciences</td>
			<td>CD511B-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge refrigerated</td>
			<td>Eppendorf</td>
			<td>model no. 5424 R, cat. no.5404000138</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4112APG</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes&#160;</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>3117-0380PK</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>2000c</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Progenitor cells (NPC)</td>
			<td></td>
			<td></td>
			<td>Provided by Dr. Jakub Godlewski (Brigham and Women&#8217;s Hospital, Boston, MA)</td>
		</tr>
		<tr>
			<td>Neurobasal Medium&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon eclipse Ti motorized fluorescent microscope system</td>
			<td>Nikon, Japan</td>
			<td>14314</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985088</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>CLS6571-960EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri-Dishes 94/16&#160;</td>
			<td>Greiner Bio-One</td>
			<td>632180</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine&#160;</td>
			<td>Sigma- Aldrich</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF&#160;</td>
			<td>PeproTech&#160;</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic&#160;</td>
			<td>PeproTech&#160;</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Gibco</td>
			<td>12587-010&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Miniprep Kit</td>
			<td>Direct-zol</td>
			<td>R2050</td>
			<td></td>
		</tr>
		<tr>
			<td>S1000 Thermal Cycler&#160;</td>
			<td>Bio-Rad</td>
			<td>1852196</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Image-Guided Micro Irradiator&#160;</td>
			<td>Xstrahal Life Sciences, UK</td>
			<td>Core facility (Dana-Farber Cancer Institute, Boston, MA)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall WX+ Ultracentrifuge&#160;</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>75000100</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOne Real-Time PCR System</td>
			<td>Applied Biosystems&#160;</td>
			<td>4376357</td>
			<td></td>
		</tr>
		<tr>
			<td>SterilGARD biosafety cabinet&#160;</td>
			<td>The Baker Company</td>
			<td>SG403A-HE</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>T98-G</td>
			<td>American Type Culture Collecti</td>
			<td>ATCC CRL-1690</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4366596</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4324018</td>
			<td></td>
		</tr>
		<tr>
			<td>Temozolomide</td>
			<td>Tocris Bioscience</td>
			<td>2706</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek optimum cutting temperature&#160;</td>
			<td>Fisher Scientific</td>
			<td>NC9636948</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td>Lysis reagent</td>
		</tr>
		<tr>
			<td>U251-MG</td>
			<td>American Type Culture Collecti</td>
			<td>ATCC HTB-17</td>
			<td></td>
		</tr>
		<tr>
			<td>U87-MG&#160;</td>
			<td>American Type Culture Collecti</td>
			<td>ATCC HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td>ViraPower Lentivector Expression system&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>K4970-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, HPLC grade</td>
			<td>Fisher</td>
			<td>W54</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of functionally associated mirnas in glioblastoma and their engineering into artificial clusters for gene therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

This method allowed characterization of a module of three miRNAs that are consistently downregulated in brain tumors, which are co-expressed specifically during neuronal differentiation (Figure 1) and involved in the tumor survival response after therapy (Figure 2). This is accomplished by regulating a complex oncogenic chromatin repressive pathway. This co-expression pattern suggested a strong synergistic activity among these th...

Watch this Scientific Journal Video about Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy at JoVE.com

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

characterization-functionally-associated-mirnas-glioblastoma-their

Research

JoVE Journal

Genetics

5.6K Views.  Brigham and Women's Hospital-Harvard Medical School. Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

Video: Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...