Name: simultaneous video-eeg-ecg monitoring to identify neurocardiac dysfunction in mouse models of epilepsy
Uploaded: 2018-01-29T15:00:00
Description: Watch this Scientific Journal Video about Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy at JoVE.com

This work was supported by Citizens United for Research in Epilepsy (grant number 35489); the National Institutes of Health (grant numbers R01NS100954, R01NS099188); and a Louisiana State University Health Sciences Center Malcolm Feist Postdoctoral Fellowship.

All experimental procedures should be carried out in accordance with the guidelines of the National Institutes of Health (NIH), as approved by your institution&#39;s Institutional Animal Care and Use Committee (IACUC). The main surgical tools needed for this protocol are shown in Figure 1.
1. Preparing Electrode for Implantation
<ol>
	<li>Place the 10-socket female nanoconnector (i.e, the electrode; Figure 2A) into a tabletop vise w...

<ol>
	<li>Fisch, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fisch and Spehlmann's EEG Primer. , (1999).</li><li>Constant, I., Sabourdin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EEG+signal:+a+window+on+the+cortical+brain+activity.">The EEG signal: a window on the cortical brain activity.</a> Paediatr. Anaesth. 22 (6), 539-552 (2012).</li><li>Mendez, O. E., Brenner, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+yield+of+EEG.">Increasing the yield of EEG.</a> J. Clin. Neurophysiol. 23 (4), 282-293 (2006).</li><li>Smith, S. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEG+in+the+diagnosis,+classification,+and+management+of+patients+with+epilepsy.">EEG in the diagnosis, classification, and management of patients with epilepsy.</a> J. Neurol. Neurosurg. Psychiatry. 76, ii2-ii7 (2005).</li><li>Bauer, G., Trinka, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonconvulsive+status+epilepticus+and+coma.">Nonconvulsive status epilepticus and coma.</a> Epilepsia. 51 (2), 177-190 (2010).</li><li>Hughes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+seizures:+a+review+of+recent+reports+with+new+concepts.">Absence seizures: a review of recent reports with new concepts.</a> Epilepsy Behav. 15 (4), 404-412 (2009).</li><li>Mostacci, B., Bisulli, F., Alvisi, L., Licchetta, L., Baruzzi, A., Tinuper, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ictal+characteristics+of+psychogenic+nonepileptic+seizures:+what+we+have+learned+from+video/EEG+recordings--a+literature+review.">Ictal characteristics of psychogenic nonepileptic seizures: what we have learned from video/EEG recordings--a literature review.</a> Epilepsy Behav. 22 (2), 144-153 (2011).</li><li>Smith, S. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEG+in+neurological+conditions+other+than+epilepsy:+when+does+it+help,+what+does+it+add?.">EEG in neurological conditions other than epilepsy: when does it help, what does it add?.</a> J. Neurol. Neurosurg. Psychiatry. 76, ii8-ii12 (2005).</li><li>Kennett, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+electroencephalography.">Modern electroencephalography.</a> J. Neurol. 259 (4), 783-789 (2012).</li><li>Thaler, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Only EKG Book You'll Ever Need. , (2012).</li><li>Becker, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+electrocardiography+interpretation.">Fundamentals of electrocardiography interpretation.</a> Anesth. Prog. 53 (2), 53-63 (2006).</li><li>Luz, E. J. S., Schwartz, W. R., C&#225;mara-Ch&#225;vez, G., Menotti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECG-based+heartbeat+classification+for+arrhythmia+detection:+A+survey.">ECG-based heartbeat classification for arrhythmia detection: A survey.</a> Comput. Methods Programs Biomed. 127, 144-164 (2016).</li><li>Bardai, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epilepsy+is+a+risk+factor+for+sudden+cardiac+arrest+in+the+general+population.">Epilepsy is a risk factor for sudden cardiac arrest in the general population.</a> PloS One. 7 (8), e42749 (2012).</li><li>Lamberts, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+prevalence+of+ECG+markers+for+sudden+cardiac+arrest+in+refractory+epilepsy.">Increased prevalence of ECG markers for sudden cardiac arrest in refractory epilepsy.</a> J. Neurol. Neurosurg. Psychiatry. 86 (3), 309-313 (2015).</li><li>Thurman, D. J., Hesdorffer, D. C., French, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sudden+unexpected+death+in+epilepsy:+assessing+the+public+health+burden.">Sudden unexpected death in epilepsy: assessing the public health burden.</a> Epilepsia. 55 (10), 1479-1485 (2014).</li><li>Zayachkivsky, A., Lehmkuhle, M. J., Dudek, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Continuous+EEG+Monitoring+in+Small+Rodent+Models+of+Human+Disease+Using+the+Epoch+Wireless+Transmitter+System.">Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System.</a> J. Vis. Exp. (101), e52554 (2015).</li><li>Bertram, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+for+Seizures+in+Rodents.">Monitoring for Seizures in Rodents.</a> Models of Seizures and Epilepsy. , 97-109 (2017).</li><li>Mishra, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scn2a+deletion+improves+survival+and+brain-heart+dynamics+in+the+Kcna1-null+mouse+model+of+sudden+unexpected+death+in+epilepsy+(SUDEP).">Scn2a deletion improves survival and brain-heart dynamics in the Kcna1-null mouse model of sudden unexpected death in epilepsy (SUDEP).</a> Hum. Mol. Genet. 26 (11), 2091-2103 (2017).</li><li>Thireau, J., Zhang, B. L., Poisson, D., Babuty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+variability+in+mice:+a+theoretical+and+practical+guide.">Heart rate variability in mice: a theoretical and practical guide.</a> Exp. Physiol. 93 (1), 83-94 (2008).</li><li>Smart, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+K(V)1.1+potassium+channel+causes+epilepsy+in+mice.">Deletion of the K(V)1.1 potassium channel causes epilepsy in mice.</a> Neuron. 20 (4), 809-819 (1998).</li><li>Glasscock, E., Yoo, J. W., Chen, T. T., Klassen, T. L., Noebels, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kv1.1+potassium+channel+deficiency+reveals+brain-driven+cardiac+dysfunction+as+a+candidate+mechanism+for+sudden+unexplained+death+in+epilepsy.">Kv1.1 potassium channel deficiency reveals brain-driven cardiac dysfunction as a candidate mechanism for sudden unexplained death in epilepsy.</a> J. Neurosci. 30 (15), 5167-5175 (2010).</li><li>Moore, B. M., Jerry Jou, ., Tatalovic, C., Kaufman, M., S, E., Kline, D. D., Kunze, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kv1.1+null+mouse,+a+model+of+sudden+unexpected+death+in+epilepsy+(SUDEP).">The Kv1.1 null mouse, a model of sudden unexpected death in epilepsy (SUDEP).</a> Epilepsia. 55 (11), 1808-1816 (2014).</li><li>Ryvlin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+mechanisms+of+cardiorespiratory+arrests+in+epilepsy+monitoring+units+(MORTEMUS):+a+retrospective+study.">Incidence and mechanisms of cardiorespiratory arrests in epilepsy monitoring units (MORTEMUS): a retrospective study.</a> Lancet Neurol. 12 (10), 966-977 (2013).</li><li>Stables, C. L., Auerbach, D. S., Whitesall, S. E., D'Alecy, L. G., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+impact+of+type-1+and+type-2+diabetes+on+control+of+heart+rate+in+mice.">Differential impact of type-1 and type-2 diabetes on control of heart rate in mice.</a> Auton. Neurosci. 194, 17-25 (2016).</li><li>Gehrmann, J., Hammer, P. E., Maguire, C. T., Wakimoto, H., Triedman, J. K., Berul, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+screening+for+heart+rate+variability+in+the+mouse.">Phenotypic screening for heart rate variability in the mouse.</a> Am. J. Physiol. Heart Circ. Physiol. 279 (2), H733-H740 (2000).</li><li>Goldman, A. M., Glasscock, E., Yoo, J., Chen, T. T., Klassen, T. L., Noebels, J. 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To obtain high quality EEG-ECG recordings that are free from artifacts, every precaution should be taken to prevent degradation or loosening of the implanted electrode and wires. As an EEG head implant becomes loose, the wire contacts with the brain will degrade leading to decreased signal amplitudes. Loose implants or poor wire contacts can also cause distortion of the electrical signals, introducing movement artifacts and background noise to the recordings. To prevent potential loosening of the head implant, apply a ge...

Electroencephalography (EEG) and electrocardiography (ECG) are powerful and widely used techniques for assessing in vivo brain and cardiac function, respectively. EEG is the recording of electrical brain activity by attaching electrodes to the scalp1. The signal recorded with non-invasive EEG represents voltage fluctuations arising from summated excitatory and inhibitory postsynaptic potentials generated mainly by cortical pyramidal neurons1,2. EEG is the most common neurodiagnostic test for evaluating and managing patients with epilepsy3,4. It is especially useful when epileptic seizures occur without obvious convulsive behavioral manifestations, such as absence seizures or non-convulsive status epilepticus5,6. Conversely, non-epilepsy related conditions that lead to convulsive episodes or loss of consciousness may be misdiagnosed as epileptic seizures without video-EEG monitoring7. In addition to its usefulness in the field of epilepsy, EEG is also widely used to detect abnormal brain activity associated with sleep disorders, encephalopathies, and memory disorders, as well as to supplement general anesthesia during surgeries2,8,9.
In contrast to EEG, ECG (or EKG as it is sometimes abbreviated) is the recording of the electrical activity of the heart10. ECGs are usually performed by attaching electrodes to the limb extremities and chest wall, which allows detection of the voltage changes generated by the myocardium during each cardiac cycle of contraction and relaxation10,11. The primary ECG waveform components of a normal cardiac cycle include the P wave, the QRS complex, and the T wave, which correspond to atrial depolarization, ventricular depolarization, and ventricular repolarization, respectively10,11. ECG monitoring is routinely used to identify cardiac arrhythmias and defects of the cardiac conduction system12. Among epilepsy patients, the importance of using ECG to identify potentially life-threatening arrhythmias is amplified since they are at significantly increased risk of sudden cardiac arrest, as well as sudden unexpected death in epilepsy13,14,15.
In addition to their clinical applications, EEG and ECG recordings have become an indispensable tool for identifying brain and heart dysfunction in mouse models of disease. Although traditionally these recordings have been performed separately, here we describe a technique to record video, EEG, and ECG simultaneously in mice. The simultaneous video-EEG-ECG method detailed here utilizes a tethered recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Historically, this tethered, or wired, &#160;configuration has been the standard and most extensively used method for EEG recordings in mice; however, wireless EEG telemetry systems have also been developed recently and are gaining in popularity16.
Compared to wireless EEG systems, the tethered arrangement possesses several technical advantages that may make it preferable depending on the desired application. These advantages include a greater number of channels for recording EEG or other biopotentials; lower electrode costs; electrode disposability; less susceptibility to signal loss; and greater frequency bandwidth (i.e., sampling rate) of recordings17. Done properly, the tethered recording method described here is capable of providing high quality, artifact-free EEG, and ECG data simultaneously, along with the corresponding video for behavioral monitoring. This EEG and ECG data can then be mined to identify neural, cardiac, or neurocardiac abnormalities such as seizures, changes in EEG power spectrum, cardiac conduction blocks (i.e., skipped heart beats), and changes in heart rate variability. To demonstrate the application of these EEG-ECG quantitative methods, we present an example experiment using a Kcna1 knockout (-/-) mouse. Kcna1-/- mice lack voltage-gated Kv1.1 &#945;-subunits and as a consequence exhibit spontaneous seizures, cardiac dysfunction, and premature death, making them an ideal model for simultaneous EEG-ECG evaluation of deleterious epilepsy-associated neurocardiac dysfunction.

<table>
	<tbody>
		<tr>
			<td>VistaVision stereozoom dissecting microscope</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dolan-Jenner MI-150 microscopy illuminator, with ring light</td>
			<td>VWR</td>
			<td>MI-150RL</td>
			<td></td>
		</tr>
		<tr>
			<td>CS Series scale</td>
			<td>Ohaus</td>
			<td>CS200</td>
			<td>for weighing animal</td>
		</tr>
		<tr>
			<td>T/Pump professional</td>
			<td>Stryker</td>
			<td></td>
			<td>recirculating water heat pad system</td>
		</tr>
		<tr>
			<td>Ideal Micro Drill</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-6300</td>
			<td></td>
		</tr>
		<tr>
			<td>Ideal Micro Drill Burr Set</td>
			<td>Cell Point Scientific</td>
			<td>60-1000</td>
			<td>only need the 0.8-mm size</td>
		</tr>
		<tr>
			<td>electric trimmer</td>
			<td>Wahl</td>
			<td>9962</td>
			<td>mini clipper</td>
		</tr>
		<tr>
			<td>tabletop vise</td>
			<td>Eclipse Tools</td>
			<td>PD-372</td>
			<td>PD-372 Mini-tabletop suction vise</td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td>ToughCut, Straight, Sharp/Sharp, 11.5 cm</td>
		</tr>
		<tr>
			<td>Crile-Wood needle holder</td>
			<td>Fine Science Tools</td>
			<td>12003-15</td>
			<td>Straight, Serrated, 15 cm, with lock - For applying wound clips</td>
		</tr>
		<tr>
			<td>Dumont #7 forceps</td>
			<td>Fine Science Tools</td>
			<td>11297-00</td>
			<td>Standard Tips, Curved, Dumostar, 11.5 cm</td>
		</tr>
		<tr>
			<td>Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td>Serrated, Straight, 12 cm</td>
		</tr>
		<tr>
			<td>Olsen-Hegar needle holder with suture cutter</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td>Straight, Serrated, 12 cm, with lock</td>
		</tr>
		<tr>
			<td>scalpel handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>surgical blades #15</td>
			<td>Havel&#39;s</td>
			<td>FHS15</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 surgical suture</td>
			<td>Unify</td>
			<td>S-N618R13</td>
			<td>non-absorbable, monofilament, black</td>
		</tr>
		<tr>
			<td>gauze sponges</td>
			<td>Coviden</td>
			<td>2346</td>
			<td>12 ply, 7.6 cm x 7.6 cm</td>
		</tr>
		<tr>
			<td>cotton-tipped swabs</td>
			<td>Constix</td>
			<td>SC-9</td>
			<td>15.2-cm total length</td>
		</tr>
		<tr>
			<td>super glue&#160;</td>
			<td>Loctite</td>
			<td>LOC1364076</td>
			<td>gel control</td>
		</tr>
		<tr>
			<td>Michel wound clips, 7.5mm</td>
			<td>Kent Scientific</td>
			<td>INS700750</td>
			<td></td>
		</tr>
		<tr>
			<td>polycarboxylate dental cement kit</td>
			<td>Prime-dent</td>
			<td>010-036</td>
			<td>Type 1 fine grain</td>
		</tr>
		<tr>
			<td>tuberculin syringe</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427431</td>
			<td>PE160, 1.143 mm (ID) x 1.575 mm (OD)</td>
		</tr>
		<tr>
			<td>chlorhexidine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C9394</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube vet ointment</td>
			<td>Dechra Veterinary Products</td>
			<td></td>
			<td>opthalamic eye ointment</td>
		</tr>
		<tr>
			<td>mouse anesthetic cocktail</td>
			<td></td>
			<td></td>
			<td>Ketamine (80 mg/kg), Xylazine (10 mg/kg), and Acepromazine (1 mg/kg)</td>
		</tr>
		<tr>
			<td>carprofen</td>
			<td></td>
			<td></td>
			<td>Rimadyl (trade name)</td>
		</tr>
		<tr>
			<td>HydroGel</td>
			<td>ClearH20</td>
			<td>70-01-5022</td>
			<td>hydrating gel; 56-g cups</td>
		</tr>
		<tr>
			<td>Ponemah&#160; software</td>
			<td>Data Sciences International</td>
			<td></td>
			<td>data acquisition and analysis software; version 5.2 or greater with Electrocardiogram Module</td>
		</tr>
		<tr>
			<td>7700 Digital Signal conditioner</td>
			<td>Data Sciences International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 Channel Isolated Bio-potential Pod</td>
			<td>Data Sciences International</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fish tank</td>
			<td>Topfin</td>
			<td></td>
			<td>for use as recording chamber; 20.8 gallon aquarium; 40.8 cm (L) X 21.3 cm (W) X 25.5 cm (H)</td>
		</tr>
		<tr>
			<td>Digital Communication Module (DCOM)</td>
			<td>Data Sciences International</td>
			<td>13-7715-70</td>
			<td></td>
		</tr>
		<tr>
			<td>12 Channel Isolated Bio-potential Pod</td>
			<td>Data Sciences International</td>
			<td>12-7770-BIO12</td>
			<td></td>
		</tr>
		<tr>
			<td>serial link cable</td>
			<td>Data Sciences International</td>
			<td>J03557-20</td>
			<td>connects DCOM to bio-potential pod</td>
		</tr>
		<tr>
			<td>Acquisition Interface (ACQ-7700USB)</td>
			<td>Data Sciences International</td>
			<td>PNM-P3P-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>network video camera</td>
			<td>Axis Communications</td>
			<td></td>
			<td>P1343, day/night capability</td>
		</tr>
		<tr>
			<td>8-Port Gigabit Smart Switch</td>
			<td>Cisco</td>
			<td>SG200-08</td>
			<td>8-port gigabit ethernet swith with 4 power over ethernet supported ports (Cisco Small Business 200 Series)</td>
		</tr>
		<tr>
			<td>10-pin male nanoconnector with guide post hole</td>
			<td>Omnetics</td>
			<td>NPS-10-WD-30.0-C-G</td>
			<td>electrode for implantation on the mouse head</td>
		</tr>
		<tr>
			<td>10-socket female nanoconnector with guide post</td>
			<td>Omnetics</td>
			<td>NSS-10-WD-2.0-C-G</td>
			<td>connector for electrode implant</td>
		</tr>
		<tr>
			<td>1.5-mm female touchproof connector cables</td>
			<td>PlasticsOne</td>
			<td>441</td>
			<td>1 signal, gold-plated; for connecting the wiring from the head-mount implant to the bio-potential pod</td>
		</tr>
		<tr>
			<td>soldering iron</td>
			<td>Weller</td>
			<td>WESD51 BUNDLE</td>
			<td>digital soldering station</td>
		</tr>
		<tr>
			<td>solder</td>
			<td>Bernzomatic</td>
			<td>327797</td>
			<td>lead free, silver bearing, acid flux core solder</td>
		</tr>
		<tr>
			<td>heat shrink tubing</td>
			<td>URBEST</td>
			<td></td>
			<td>collection of tubing with 1.5- to 10-mm internal diameters</td>
		</tr>
		<tr>
			<td>heat gun</td>
			<td>Dewalt</td>
			<td>D26960</td>
			<td></td>
		</tr>
		<tr>
			<td>mounting tape (double-sided)</td>
			<td>3M Scotch</td>
			<td>MMM114</td>
			<td>114/DC Heavy Duty Mounting Tape, 2.54 cm x 1.27 m&#160;</td>
		</tr>
		<tr>
			<td>desktop computer</td>
			<td>Dell</td>
			<td></td>
			<td>recommended minimum requirements: 3rd Gen Intel Core i7-3770 processor with HD4000 graphics; 4 GB RAM, 1 GB AMD Radeon HD 7570 video card; 1 TB hard drive; Windows 7 OS&#160;</td>
		</tr>
		<tr>
			<td>permanent marker</td>
			<td>Sharpie</td>
			<td>37001</td>
			<td>black color, ultra fine point</td>
		</tr>
		<tr>
			<td>toothpicks</td>
			<td></td>
			<td></td>
			<td>for mixing and applying the polycarboxylate dental cement</td>
		</tr>
		<tr>
			<td>LabChart Pro software</td>
			<td>ADInstruments</td>
			<td></td>
			<td>power spectrum software; version 8.1.3 or greater</td>
		</tr>
		<tr>
			<td>Kubios HRV software</td>
			<td>Univ. of Eastern Finland</td>
			<td></td>
			<td>HRV analysis software; version 2.2</td>
		</tr>
		<tr>
			<td>Notepad</td>
			<td>Microsoft</td>
			<td></td>
			<td>simple text editor software</td>
		</tr>
	</tbody>
</table>

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.

To demonstrate how to analyze the data from EEG-ECG recordings to identify neurocardiac abnormalities, results are shown for a 24-h EEG-ECG recording of a Kcna1-/- mouse (2 months old). These mutant animals, which are engineered to lack voltage-gated Kv1.1 &#945;-subunits encoded by the Kcna1 gene, are a frequently used genetic model of epilepsy since they exhibit reliable and frequent generalized tonic-clonic seizure activity beginning a...

Watch this Scientific Journal Video about Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy at JoVE.com

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

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 ...

The overall goal of this procedure is to measure brain and heart biosignals in mice using simultaneous recordings of video, electroencephalography, and electrocardiography. This method can help answer key questions in the field of epilepsy, such as does a gene mutation in mice cause spontaneous seizures and do seizures evoke cardiac arrhythmias. The main advantage of this technique is that the tethered recording configuration allows more biopotential recording channels than wireless systems and it utilizes electrodes that are relatively inexpensive and disposable.So this method can provide insight in cardiac dysfunction in epilepsy, it can also be applied to the study of neurocardiology to identify associations between arrhythmias and abnormal brain activity. To begin this procedure place the 10 socket female nano connector into a tabletop vice with the 10 wires facing up and the black wire in the front. Using fine forceps fold down the black wire to the right and the tan wire to the left.Next fold down the red, orange, blue, and purple wires alternating right and left. Afterward, cut off the yellow, green, white, and gray wires at the base of their attachment. To prepare the ECG wires, use a permanent marker to make marks on the purple wire at 3.2 centimeters and 3.5 centimeters from the base of the electrode and on the blue wire at 2.2 centimeters and 2.5 centimeters.Remove the electrode from the vice and expose the silver filaments between the marked areas by stripping the insulation on side of the wire with a scalpel blade. Subsequently, place the electrode back in the vice. Affix a piece of double sided mounting tape, which has be precut to the length and width of the electrode to the top of the wires using a thin layer of superglue.Then trim the wires to be used for EEG at a slightly V-shaped angle to a length of approximately seven to nine millimeters, with the tan and black wires cut to the shortest. Keep in mind not to cut the wires to be used for ECG. In this procedure, shave two small areas about two square centimeters on both sides of the trunk of the mouse corresponding to where the ECG wires will be implanted.Next, place the mouse in the prone position on the stage of the dissecting microscope and confirm the adequate depth of anesthesia by the absence of the toe pinch reflex. Holding the head steady between the thumb and forefinger, part the fur down the center of the head from between the ears to just behind the eyes with a cotton swab soaked I alcohol. Using a scalpel make a one centimeter midline incision through the scalp between the parted fur, from just in front of the ears to the just between the eyes.Using either the side of the scalpel or a cotton tip applicator, gently scrape the mucus membrane on top of the skull until the bone appears dry. Then pluck the fur around the perimeter of the incision forming a thin border of bald skin. Carefully remove any fur that may have fallen into the surgical field with a pair of forceps.Dry the surface of the skull with a cotton tip applicator applying gentle pressure for several seconds if needed. Following that, make four marks on the skull with a permanent marker at the sites where the burr holes will be drilled. Place two marks, one on each side of the sagittal suture, anterior to bregma for the reference and ground wires.Subsequently place another two marks, one one each side of the sagittal suture posterior to bregma for the two EEG recording wires. Using a microdrill, make small burr holes at each mark with a 0.8 millimeter diameter drill bit. Apply gentle pressure while drilling to create small recesses at each marked spot.Drill through the skull by pulsing the drill bit as the hole nears completion, being sure not to apply too much pressure, which could lead to penetrating and damaging the underlying brain tissue. After all holes are drilled, wipe the area clean with a cotton tip applicator. To adhere the electrode to the top of the skull, remove the paper backing from the double sided mounting tape on the electrode.Apply a thin layer of superglue to the tape. Using a pair of forceps, remove the electrode from the vice. Orient the electrode such that when positioned along the sagittal suture, the shorter EEG wires are rostral and the longer ECG wires are caudal.Adhere the electrode to the skull over the sagittal suture between the holes. To implant the wires for ECG, rotate the mouse slightly onto its right side, while keeping the head upright. Take the long ECG wire on the left side and extend it down the side of the mouse to the shaved area on the left side.Visualize where the exposed wire will be positioned once it is tunneled beneath the skin. Then using a scalpel, make a on centimeter incision in the skin at the location where the exposed wire will be positioned. While holding the incision open, use forceps to loosen the skin around the incision from the underlying connective tissue to form a pocket for the wire.Beginning at the incision site on the side of the animal tunnel subcutaneously with a piece of six centimeter long polyethylene tubing with a beveled leading edge until the beveled edge exits the incision made on the head. Feed the ECG wire through the tubing. While removing the tubing, grasp the electrode wire as it exits the lateral incision and pull the wire taut.Next, fix the ECG wire in place by suturing it to the tissue under the skin. Apply one suture over the exposed filaments and another suture either before or after the exposed portion. Cut the electrode wire about two to three millimeters past the last suture and tuck the end into to the pocket of skin formed previously.After that, pull two sides of the incision together and close with a wound clip. Repeat the above steps to place the contralateral ECG wire. To implant the wires for EEG, starting with the most anterior hole on one side, bend the wire that is closest to that hole so that it is positioned directly over the hole, but not yet inserted.Grasp the lower end of the wire and feed it as horizontally as possible into the hole until two to three millimeters of the wire is under the skull. With the end of the wire secured in the hole gently fold down the remaining portion of the wire so that it lies flat against the skull. Continue in the same manner with the posterior wire on the same side.Repeat the procedures for the anterior and posterior wires on the other side. In this procedure, mix two scoops of polycarboxylate powder with five drops of polycarboxylate liquid. Stir the mixture with a toothpick to make a paste with the desired viscosity.Then pick up a large drop of cement with the toothpick and apply around the base of the electrode beginning caudally. Continue around the electrode allowing the cement to drip over the wires, forming a cap around the implant. Using to Dumont forceps, pull the fur at the edges of the incision up over the cement cap and press together, being careful not to disturb the wires implanted beneath.Subsequently press the fur up into the cement to help with closure. After a recovery period, tether the mouse by gently but firmly holding the mouse in one hand while using the other hand to insert the 10 pin metal connector with guidepost into the sockets of the EEG, ECG electrode implant on the mouse's head. Then transfer the mouse to a recording chamber with transparent walls to facilitate video monitoring.Record simultaneous video and EEG, ECG and save the digitized data for offline analyses with signal processing software. Shown here is an EEG trace representing spontaneous seizure in a KCNA1 knockout mouse. This is a plot of the time durations of each seizure observed during the 24 hour recording session in the mouse.And this spectrogram shows the frequency and power density before, during, and after a seizure. Comparison of the relative power in each EEG frequency band during the pre and post-ictal periods reveals an increase in relative delta power and decreases in theta, alpha, beta, and gamma power. And this is a representative plot of an R-R interval series obtained from the ECG recording of the KCNA1 knockout mouse, showing the fluctuations in the time between beats.The red line shows the low frequency trend components that give removed from the R-R interval series following detrending. Here is a sample ECG trace from a KCNA1 knockout mouse showing normal sinus rhythm preceding an atrioventricular conduction block, which manifests as a P-wave that is not followed by QRS complex. Once mastered, this surgery can be done in 50 minutes if it is performed properly.After watching this video, you should have a good understanding of how to implant electrodes for recording electroencephalography and electrocardiography in mice, which can be applied in epilepsy models to identify neurocardiac dysfunction.

simultaneous-video-eeg-ecg-monitoring-to-identify-neurocardiac

Research

JoVE Journal

Genetics

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...