Name: viral transgene expression in rodent hearts and the assessment of cardiac arrhythmia risk
Uploaded: 2022-07-27T14:10:00
Description: Watch this Scientific Journal Video about Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk at JoVE.com

This work was supported by the Canadian Institutes of Health Research (CIHR) Project Grants (PJT-148918 and PJT-180533, to WL), the CIHR Early Career Investigator Award (AR8-162705, to WL), the Heart and Stroke Foundation of Canada (HSFC) McDonald Scholarship and New Investigator Award (S-17-LI-0866, to WL), Student Scholarships (to JW and YX), and a Postdoctoral Fellowship (to AL) from the University of Ottawa Cardiac Endowment Funds at the Heart Institute. The authors thank Mr. Richard Seymour for his technical support. Figure 2 was created with Biorender.com with approved licenses.

All the methods and procedures described were approved by the animal research ethical review board at the University of Ottawa and the biosafety review committee at the University of Ottawa Heart Institute. The developed safety protocols include that all the procedures dealing with recombinant adenovirus or adeno-associated virus (AAV) were performed in a level II biosafety cabinet. All the items in contact with the virus were thoroughly decontaminated after the experiment. Ctnnb1flox/flox and &#945;M...

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Several steps are critical for the success of the Langendorff-perfused, isolated heart preparation. Firstly, it is important to avoid any damage to the heart during heart collection (e.g., due to accidental squeezing or cutting with the scissors). Secondly, it is critical to put the collected heart into cold Tyrode solution as soon as possible because this will stop the heartbeat and reduce the oxygen consumption of the heart. Thirdly, the needle insertion into the aorta must not be too deep&#8212;ideally, the tip of the...

Cardiovascular disease is the leading cause of death worldwide, claiming the lives of 18 million people in 2017 alone1. Rodents, especially mice and rats, have become the most commonly used model in cardiovascular research due to the ease of handling and the availability of various transgenic overexpression or knockout lines. Rodent models have been fundamental for understanding the disease mechanisms and for identifying potential new therapeutic targets in myocardial infarction2, hypertension3, heart failure4, and atherosclerosis5. However, the use of rodents in studies of cardiac arrhythmias is limited by their small heart size and faster heart rate compared to human or large animal models. Therefore, spontaneous lethal arrhythmias in mice or rats after myocardial infarction are rare2. Investigators are forced to focus on indirect secondary changes that might reflect a pro-arrhythmic substrate, such as fibrosis or gene expression, without showing meaningful changes in arrhythmia burden or pro-arrhythmic tendencies. To overcome this limitation, a method that allows a reliable assessment of the susceptibility of mouse and rat hearts to ventricular tachyarrhythmias after genetic modification6,7 or myocardial infarction2 is described in the present protocol. This method combines adrenergic receptor stimulation with programmed electrical stimulation to induce ventricular tachyarrhythmias in isolated, Langendorff-perfused8 mouse and rat hearts.
Standard approaches for viral gene transfer in rodent myocardial tissue often involve the exposure of the heart by thoracotomy9,10,11, which is an invasive procedure and is associated with delayed recovery of the animals after the procedure. This article describes a method of direct intramyocardial injection of&#160;virus under ultrasound imaging guidance for the overexpression of transgenes. This less invasive procedure allows for faster animal recovery after viral injection and less tissue injury, as compared to thoracotomy, reduces post-operative pain and inflammation in the animal, and, thus, allows better assessment of the effects of&#160;transgenic genes on heart function.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30 G 1/2 PrecisionGlide Needle</td>
			<td>Becton Dickinson (BD)</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>adeno-associated virus (AAV9-GFP)</td>
			<td>Vector Biolabs</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>adenovirus (Ad-GFP)</td>
			<td>Vector Biolabs</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>adenovirus (Ad-Wnt3a)</td>
			<td>Vector Biolabs</td>
			<td>ADV-276318</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet (Level II)</td>
			<td>Microzone Corporation</td>
			<td>N/A</td>
			<td>Model #: BK-2-4</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Vetergesic</td>
			<td>DIN 02342510</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>102378</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher Chemical</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td>WAHL Clipper Corporation</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Sigma-Aldrich</td>
			<td>20701</td>
			<td>705 LT, volume 50 &#956;L</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Life Brand</td>
			<td>E12107</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fresenius Kabi</td>
			<td>DIN 02264315</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fresenius Kabi Ltd.</td>
			<td>M60303</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproterenol hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>1351005</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart 8 software</td>
			<td>ADInstruments Inc.</td>
			<td>Version 8.1.5</td>
			<td>for ECG recording</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice (Ctnnb1flox/flox)</td>
			<td>Jackson Labs</td>
			<td>4152</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice (&#945;MHC-MerCreMer)</td>
			<td>Jackson Labs</td>
			<td>5650</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>S9i</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>MS400 transducer</td>
			<td>VisualSonic Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Systane</td>
			<td>DIN 02444062</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure meter</td>
			<td>NETECH</td>
			<td>DigiMano 1000</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>Cole-Parmer</td>
			<td>UZ-77924-65</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Rat (Sprague-Dawley, male)</td>
			<td>Charles River</td>
			<td>400</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10007-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomer</td>
			<td>Down Inc.</td>
			<td>Sylgard 184</td>
			<td>for Langendorff system</td>
		</tr>
		<tr>
			<td>Small animal ECG system</td>
			<td>ADInstruments Inc.</td>
			<td>N/A</td>
			<td>Powerlab 8/35 and Animal Bio Amp</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>567530</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>IonOptix</td>
			<td>MyoPacer EP</td>
			<td></td>
		</tr>
		<tr>
			<td>VEVO3100 Preclinical Imaging System</td>
			<td>VisualSonic Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

viral transgene expression in rodent hearts and the assessment of cardiac arrhythmia risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

When perfused following the protocol described here (Figure 1), an isolated rat or mouse heart beats rhythmically and stably for at least 4 h. If the experimental design requires a longer period of heart perfusion, it is helpful to add albumin into the perfusion solution to reduce the occurrence of myocardial edema after long-time perfusion14. The inclusion of isoproterenol in the perfusion solution mimics the activation of the sympathetic nervous system&#160;which oc...

Watch this Scientific Journal Video about Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk at JoVE.com

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

This protocol allows the user to achieve efficient transgene expression in the ventricular myocardium, facilitating gene expression studies to examine cardiac arrhythmias and function in the ventricular tissue. The technique for viral transgene expression is minimally invasive for the animal, causing less damage and reduced recovery time than conventional approaches that involve a thoracotomy. The technique for inducing arrhythmias in small rodent hearts is useful for assessing the susceptibility to arrhythmias in small animal models of heart disease.Demonstrating the procedure will be Doctor Alice Lu, a post-doctoral fellow from the lab. To begin, sterilize the left lower chest region of the anesthetized mouse with alternating rounds of an iodine-based or chlorhexidine-based scrub, in alcohol, three times in a circular motion. Under ultrasound imaging guidance, insert the needle of the syringe containing the virus into the animal's chest.Approach the needle tip into the left ventricular front free wall and slowly inject 10 to 15 microliters of the virus. Verify successful injection in ultrasound images by the enhanced brightness near the tip of the needle. Withdraw the needle from the heart and insert into other regions of the left ventricle for a second and third injection of the same amount of virus.To perform Langendorff perfusion of the mouse heart, place the cannulated heart in a silicone-elastomer-coated, 10-centimeter plastic dish, with the left ventricle facing up. Cannulate the aorta of the heart with a blunt needle connected to a modified Langendorff profusion system in the constant flow rate mode. Perfuse the heart with oxygen-bubbled Tyrode solution at 37 degrees Celsius.At the beginning of perfusion, verify the correct aortic cannulation by observing the washout of blood from the heart during the first two to three heartbeats and changing the heart color from red to pale. Adjust the flow rate to keep the perfusion pressure at 70 to 80 millimeters of mercury. After confirmation, place the electrodes of a small animal ECG system around the heart by inserting them into the silicone-elastomer coating in the dish.Then, record the ECG using compatible software. Next, perform adrenergic receptor stimulation and perfuse the heart with isoproterenol and Tyrode solution. After 10 minutes, perform programmed electrical stimulation to induce ventricular tachyarrhythmias by stimulating the heart at the apex with two platinum electrodes connected to an electrical stimulator.Start the stimulation procedure with initial 10 consecutive stimuli, S1, followed by an extra stimulus, S2, with an initial interval of 80 milliseconds. Then, repeatedly reduce the S2 interval by two milliseconds each time until the heartbeat can no longer be captured or the heart's effective refractory period, or ERP, is reached. Monitor any induced ventricular tachyarrhythmias, including ventricular tachycardia and fibrillation, by ECG.If no arrhythmias are induced. add another extra stimulus, S3, after S2, with the same parameters until the ERP is reached. If ventricular tachyarrhythmias are still not induced, stop the electrical stimulation and consider the heart non-inducible.During the programmed electrical stimulation, the successful pacing of the heart is verified by the one-to-one capture of the heartbeat during the consecutive S1 stimuli and the prolonged QRS complex during S1 pacing. These combined adrenergic and electrical stimulations induced no ventricular tachyarrhythmias in healthy, sham-operated wild-type mouse hearts, or in control adenovirus-GFP-injected rat hearts. In contrast, the same protocol induced ventricular tachyarrhythmias in 77%of wild-type mouse hearts after myocardial infarction and in three out of four rats after intramyocardial injection of Ad-Wnt3a.While inserting the needle, ensure that the needle tip is embedded within the free wall and not inside the ventricle. Also, securing and cannulating the aorta of the heart is the most critical step. Following these procedures, hearts can be used for standard histological studies or molecular biology assays.Alternatively, live cells can be isolated in order to perform single-cell electrophysiology studies.

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Biology

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.

This protocol describes a method to inject plasmid DNA into the mouse kidney via the renal pelvis to produce transgene expression specifically in the kidney.

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...