This study was carried out in the Center for the Study of Mitochondrial Pediatric Diseases (http://www.mitopedia.org), funded by the Mariani Foundation. VT is a member of the European Reference Network for Rare Neuromuscular Diseases (ERN EURO-NMD).

NOTE: The use of human fibroblasts may require ethical approval. Fibroblasts used in this study were derived from MD patients and stored in the Institutional biobank in compliance with ethical requirements. Informed consent was provided for the use of the cells. Perform all cell culture procedures under a sterile laminar flow cabinet at room temperature (RT, 22-25 &#176;C). Use sterile-filtered solutions suitable for cell culture and sterile equipment. Grow all cell lines in a humidified incubator at 37 &#176;C with 5% CO2. Mycoplasma tests should be conducted weekly to ensure mycoplasma-free cultures. 143BTK- rho0 cells can be generated as previously described5.
1. Culture of cells
<ol>
	<li>Before starting any procedure, verify the presence of the mutation in fibroblasts derived from MD patient(s) and quantify the percentage of heteroplasmy or homoplasmy by Restriction Fragment Length Polymorphism (RFLP) and/or whole mtDNA sequencing analyses14.</li>
	<li>Seed fibroblasts in four 35 mm Petri dishes, each containing 2 mL of Complete Culture Medium (Table 1). Let the cells grow until 80% confluent (48 h).</li>
	<li>Grow 143BTK- rho0 cells in 8 mL of Supplemented&#160;Culture Medium (Table 1) in a 100 mm Petri dish.</li>
	<li>Maintain the cells in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>Check the absence of mtDNA in rho0 cells by sequencing techniques14.</li>
</ol>
2. Enucleation of fibroblasts
<ol>
	<li>Sterilize four 250 mL centrifuge-suitable bottles by autoclave sterilization at 121 &#176;C for a 20 min cycle. Dry them in a laboratory oven or at RT.</li>
	<li>Prewarm the centrifuge at 37 &#176;C.</li>
	<li>Wash the 35 mm dishes containing the fibroblasts twice, using 2 mL of 1x phosphate-buffered saline (PBS) without (w/o) calcium and magnesium.</li>
	<li>Clean the outer surface of the dishes with 70% ethanol and wait until the alcohol evaporates.</li>
	<li>Remove the lids from the dishes and the screw caps from the bottles. Place each dish, without the lid, upside down on the bottom of each 250 mL centrifuge bottle.</li>
	<li>Slowly add 32 mL of Enucleation Medium to each bottle (Table 1), allowing the medium to enter the dish and come into contact with the cells. Remove any bubbles from the dishes using a long glass Pasteur pipette, curving the tip in a Bunsen flame. 
	NOTE: It is important to remove the bubbles to allow the medium to enter the dish and come in contact with the cells.</li>
	<li>Close each bottle with the screw cap and transfer them to the centrifuge.</li>
	<li>Centrifuge for 20 min at 37 &#176;C and 8,000 &#215; g, acceleration max, deceleration slow. Pay attention to balance the centrifuge: counterweight each bottle. If necessary, adjust the weight by adding a suitable volume of Enucleation Medium.</li>
	<li>During centrifugation, use vacuum or a 10 mL serological pipette to aspirate and discard the medium from the 143BTK- rho0 culture plates and wash them twice using 4 mL of 1x PBS w/o calcium and magnesium.</li>
	<li>Add 2 mL of trypsin to cover the cell monolayer completely.</li>
	<li>Place the dishes in a 37 &#176;C incubator for ~2 min.</li>
	<li>Remove the dishes from the incubator, observe cell detachment using an inverted microscope for live cells (objectives 4x or 10x), and inhibit the enzyme activity by adding 2 mL of Supplemented Culture Medium.</li>
	<li>Aspirate the 4 mL of the cell suspension in the dish with a 10 mL pipette and transfer it to a 15 mL conical tube.</li>
	<li>Count the cells using a Burker hemocytometer chamber or an automated counter.</li>
	<li>At the end of centrifugation (step 2.8), aspirate the medium from the bottles and discard it.</li>
	<li>Remove the dishes by inverting the bottles on a sterile gauze previously sprayed with 70% ethanol. Clean the outer surface of the dishes and their lids with 70% ethanol. Wait until the alcohol evaporates, and then close the dishes.</li>
	<li>Before proceeding, check for cytoplast (ghost) formation using an inverted microscope for live cells (objective 4x or 10x). Look for extremely elongated fibroblasts due to the extrusion of their nuclei induced by cytochalasin B.</li>
	<li>To each 35 mm dish, add 1 &#215; 106 of 143BTK- rho0 cells resuspended in 2 mL of 143BTK- rho0 culture medium supplemented with 5% fetal bovine serum (FBS).</li>
	<li>Leave the dishes for 3 h in a humidified incubator at 37 &#176;C and 5% CO2 and let the 143BTK- rho0 cells settle on the ghosts. Do not disturb the dishes.</li>
</ol>
3. Fusion of the enucleated fibroblasts with rho&#160;0 cells
<ol>
	<li>After 3 h of incubation, aspirate and discard the medium from the dishes.</li>
	<li>Wash the adherent cells twice with 2 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose w/o serum or with Minimum Essential Medium (MEM).</li>
	<li>Aspirate and discard the medium.</li>
	<li>Add 500 &#181;L of PEG solution (see the Table of Materials) to the cells and incubate for exactly 1 min.</li>
	<li>Aspirate and discard the PEG solution.</li>
	<li>Wash the cells three times using 2 mL of DMEM high glucose w/o serum or with MEM.</li>
	<li>Add 2 mL of Fusion Medium (Table 1) and incubate overnight in the incubator at 37 &#176;C with 5% CO2.</li>
</ol>
4. Cybrid selection and expansion
<ol>
	<li>After overnight incubation, remove the plates from the incubator, trypsinize the cells as described above (steps 2.9-2.13), and transfer the content of each 35 mm dish into a 100 mm dish.</li>
	<li>Add 8 mL of Selection Medium (Table 1) and place the plates in the incubator at 37 &#176;C with 5% CO2.</li>
	<li>Change the medium every 2-3 days.</li>
	<li>Wait for ~10-15 days of selection until colonies of cells appear.</li>
	<li>Freeze one of the four Petri dishes by collecting all the clones and generating a &#34;massive&#34; culture as a backup of the cybrids, which can be eventually recloned and used for further investigations.</li>
	<li>Trypsinize the cells in the remaining culture dishes, count, and seed into one or more Petri dishes at 50-100 cells/dish in the Supplemented Culture Medium (Table 1) until clones appear. Let them grow for some days.</li>
	<li>Pellet the remaining cells by centrifugation at 1,200 &#215; g for 3 min at RT and discard the supernatant.</li>
	<li>Extract DNA from the pellet (see the Table of Materials).</li>
	<li>Perform genotyping by variable number of tandemly repeated (VNTR) analysis as previously reported15.</li>
	<li>Pick up clones from the Petri dish with cloning cylinders or a pipette tip, using a stereomicroscope to avoid pooling of different clones, and transfer them to a 96-well plate, each well containing 200 &#181;L of Supplemented Culture Medium (Table 1).</li>
	<li>Expand every clone until there are enough cells for freezing and extracting DNA.</li>
	<li>Verify the mutation percentage of each clone by RFLP or other sequencing methods. Ideally, try to obtain both clones with wild-type mtDNA (0% mutation) and clones with different mutation percentages, both adding up to homoplasmic mutant mtDNA (100% mutation). See Figure 1 for a schematic diagram of the cybrid generation protocol.</li>
</ol>

<ol>
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The authors have no conflicts of interest to disclose.

The mtDNA has a very high mutation rate compared to nDNA because of the lack of protective histones and its location close to the respiratory chain, which exposes the molecule to damaging oxidative effects not efficiently counteracted by the repair systems16. The first pathogenic mtDNA mutations were identified in 198817,18, and since then, a large number of mutations have been described. NGS technology is a relevant approach to screen the...

Mitochondrial disorders (MDs) are a group of multisystem syndromes caused by an impairment in mitochondrial functions due to mutations in either nuclear (nDNA) or mitochondrial (mtDNA) DNA1. They are among the most common inherited metabolic diseases, with a prevalence of 1:5,000. mtDNA-associated diseases follow the rules of mitochondrial genetics: maternal inheritance, heteroplasmy and threshold effect, and mitotic segregation2. Human mtDNA is a double-stranded DNA circle of 16.6 kb, which contains a short control region with sequences needed for replication and transcription, 13 protein-coding genes (all subunits of the respiratory chain), 22 tRNA, and 2 rRNA genes3.
In healthy individuals, there is one single mtDNA genotype (homoplasmy), whereas more than one genotype coexists (heteroplasmy) in pathological conditions. Deleterious heteroplasmic mutations must overcome a critical threshold to disrupt OXPHOS and cause diseases that can affect any organ at any age4. The dual genetics of OXPHOS dictates inheritance: autosomal recessive or dominant and X-linked for nDNA mutations, maternal for mtDNA mutations, plus sporadic cases both for nDNA and mtDNA.
At the beginning of the mitochondrial medicine era, a landmark experiment by King and Attardi5 established the basis to understand the origin of a mutation responsible for an MD by creating hybrid cells containing nuclei from tumor cell lines in which mtDNA was entirely depleted (rho0 cells) and mitochondria from patients with MDs. Next-generation sequencing (NGS) techniques were not available at that time, and it was not easy to determine whether a mutation was present in the nuclear or mitochondrial genome. The method, described in 1989, was then used by several researchers working in the field of mitochondrial medicine6,7,8,9; a detailed protocol has been recently published10, but no video has been made yet. Why should such a protocol be relevant nowadays when NGS could precisely and rapidly identify where a mutation is located? The answer is that cybrid generation is still the state-of-the-art protocol to understand the pathogenic role of any novel mtDNA mutation, correlate the percentage of heteroplasmy with the severity of the disease, and perform a biochemical investigation in a homogeneous nuclear system in which the contribution of the autochthonous nuclear background of the patient is absent11,12,13.
This protocol describes how to obtain cytoplasts from confluent, patient-derived fibroblasts grown in 35 mm Petri dishes. Centrifugation of the dishes in the presence of cytochalasin B allows the isolation of enucleated cytoplasts, which are then fused with rho0 cells in the presence of polyethylene glycol (PEG). The resulting cybrids are then cultivated in selective medium until clones arise. The representative results section shows an example of molecular characterization of the resulting cybrids to prove that the mtDNA is identical to that of the donor patients&#39; fibroblasts and that the nDNA is identical to the nuclear DNA of the tumoral rho0 cell line.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-Bromo-2&#39;-Deoxyuridine</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>B5002-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well Plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Plates</td>
			<td>Corning</td>
			<td>3596</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood and Cell Culture DNA extraction kit</td>
			<td>QIAGEN</td>
			<td>13323</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Avanti J-25</td>
			<td>7,200 rcf, 37 &#176;C</td>
		</tr>
		<tr>
			<td>Centrifuge bottles, 250 mL</td>
			<td>Beckman Coulter</td>
			<td>356011</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin B from Drechslera dematioidea</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>C2743-200UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialyzed FBS</td>
			<td>Gibco</td>
			<td>26400-036 100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM High Glucose (w/o L-Glutamine W/Sodium Pyruvate)</td>
			<td>EuroClone</td>
			<td>ECB7501L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline - PBS (w/o Calcium w/o Magnesium)</td>
			<td>EuroClone</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol Absolute Anhydrous</td>
			<td>Carlo Erba</td>
			<td>414601</td>
			<td></td>
		</tr>
		<tr>
			<td>FetalClone III (Bovine Serum Product)</td>
			<td>Cytiva - HyClone Laboratories</td>
			<td>SH30109.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pasteur pipettes</td>
			<td>VWR</td>
			<td>M4150NO250SP4</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Research Microscope For Live Cell Microscopy</td>
			<td>Nikon</td>
			<td>ECLIPSE TE200</td>
			<td></td>
		</tr>
		<tr>
			<td>JA-14 Fixed-Angle Aluminum Rotor</td>
			<td>Beckman Coulter</td>
			<td>339247</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory autoclave Vapormatic 770</td>
			<td>Labotech</td>
			<td>29960014</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (100x)</td>
			<td>EuroClone</td>
			<td>ECB 3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium MEM</td>
			<td>Euroclone</td>
			<td>ECB2071L</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td>LT07-318</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG (Polyethylene glicol solution)</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>P7181-5X5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (solution 100x)</td>
			<td>EuroClone</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>Primo TC Dishes 100 mm</td>
			<td>EuroClone</td>
			<td>ET2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Primo TC Dishes 35 mm</td>
			<td>EuroClone</td>
			<td>ET2035</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate 100 mM</td>
			<td>EuroClone</td>
			<td>ECM0542D</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 2.5% in HBSS</td>
			<td>EuroClone</td>
			<td>ECB3051D</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich (Merck)</td>
			<td>U3003-5G</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro approach to study mitochondrial dysfunction: a cybrid model

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes.
The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called &#34;transmitochondrial cybrids.&#34; Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

Generating cybrids requires 3 days of laboratory work plus a selection period (~2 weeks) and additional 1-2 weeks for the growth of clones. The critical steps are the quality of cytoplasts and the selection period. The morphology of cybrids resembles that of the rho0 donor cells. Assignment of the correct mtDNA and nDNA in the cybrids is mandatory to confirm the identity of the cells. An example is given in Figure 2. In this case, we generated cybrids starting from fibroblasts der...

Watch this Scientific Journal Video about An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model at JoVE.com

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Transmitochondrial cybrids are hybrid cells obtained by fusing mitochondrial DNA (mtDNA)-depleted cells (rho0 cells) with cytoplasts (enucleated cells) derived from patients affected by mitochondrial disorders. They allow the determination of the nuclear or mitochondrial origin of the disease, evaluation of biochemical activity, and confirmation of the pathogenetic role of mtDNA-related variants.

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human ...

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Genetics

3.6K Views.  Fondazione IRCCS Istituto Neurologico Carlo Besta. Transmitochondrial cybrids are hybrid cells obtained by fusing mitochondrial DNA (mtDNA)-depleted cells (rho0 cells) with cytoplasts (enucleated cells) derived from patients affected by mitochondrial disorders. They allow the determination of the nuclear or mitochondrial origin of the disease, evaluation of biochemical activity, and confirmation of the pathogenetic role of mtDNA-related variants.

Video: An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)&#8212;we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-&#954;B pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers&#8212;as it portends a significant roadblock against immunotherapy&#8212;the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...