Name: electroporation of plasmid dna into mouse skeletal muscle
Uploaded: 2022-04-06T15:00:00
Description: Watch this Scientific Journal Video about Electroporation of Plasmid DNA into Mouse Skeletal Muscle at JoVE.com

All experiments using animals were performed at the Penn State College of Medicine, approved by Penn State University&#39;s Institutional Animal Care and Use Committee, and performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. 12-week-old female C57BL/6 mice were used for this procedure. All surgical tools were autoclaved for sterility prior to experimentation.
1. TA and EDL injection/electroporation preparation</strong...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp27+inhibits+IKK&#946;-induced+NF-&#954;B+activity+and+skeletal+muscle+atrophy.">Hsp27 inhibits IKK&#946;-induced NF-&#954;B activity and skeletal muscle atrophy.</a> The FASEB Journal. 23 (10), 3415-3423 (2009).</li><li>Hain, B. A., Dodd, S. L., Judge, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IkappaBalpha+degradation+is+necessary+for+skeletal+muscle+atrophy+associated+with+contractile+claudication.">IkappaBalpha degradation is necessary for skeletal muscle atrophy associated with contractile claudication.</a> American Journal of Physiology Regulatory, Integregrative and Comparative Physiology. 300 (3), 595-604 (2011).</li><li>Houston, F. 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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=REDD1+deletion+attenuates+cancer+cachexia+in+mice.">REDD1 deletion attenuates cancer cachexia in mice.</a> Journal of Applied Physiology. 131 (6), 1718-1730 (2021).</li><li>Hain, B. A., Xu, H., Wilcox, J. R., Mutua, D., Waning, 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=Chemotherapy-induced+loss+of+bone+and+muscle+mass+in+a+mouse+model+of+breast+cancer+bone+metastases+and+cachexia.">Chemotherapy-induced loss of bone and muscle mass in a mouse model of breast cancer bone metastases and cachexia.</a> Journal of Cachexia, Sarcopenia and Muscle Rapid Communications. 2 (1), (2019).</li><li>Waning, D. 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In vivo gene transfer in skeletal muscle enhanced by electroporation is a useful and relatively simple tool for modulating protein expression in muscle. We have shown the steps required to achieve efficient gene transfer in the EDL and TA muscles and demonstrated that contractility measurement of the EDL is viable following the procedure. This technique does not require more complicated viral vectors and allows for the comparison of transfected and non-transfected muscle fiber cross-sectional area in a single mu...

Plasmid DNA electroporation into skeletal muscle in vivo is an important tool for assessing changes in skeletal muscle physiology and molecular signaling by modulating gene expression in a variety of physiological and pathophysiological conditions1,2,3,4,5,6,7,8,9. Experimental gene transfer into skeletal muscle was demonstrated as early as 1990 by Wolff et al., where both RNA and DNA were successfully transferred without electroporation, and luciferase expression was maintained for at least 2 months10. The relatively low transfection efficiency with injection only is problematic, and Aihara and Miyazaki demonstrated increased gene transfer with electroporation in 1998 by electroporating a pCAGGS-IL-5 construct into the tibialis anterior (TA) muscle and measuring serum IL-5 expression11. Since that time, many studies have investigated the efficacy of different DNA concentrations, volumes, and electroporation parameters to ensure maximal gene transfer efficiency. Mir et al. tested different electroporation parameters, including voltage, pulse number, pulse duration, and frequency, as well as DNA concentration, and determined that greater voltage, pulse number, and DNA concentration all contributed to increased electroporation efficiency12. A major caveat to high electroporation voltage is that, while it facilitates increased DNA uptake into myofibers, it also causes muscle damage, which can confound results. Schertzer et al. showed that electroporation at 200 V caused damage in around 50% of myofibers 3 days after electroporation, whereas only 10% of myofibers were damaged at 50 V13. We have taken into consideration the variables affecting efficient DNA transfer versus muscle damage and found that a voltage of 125 V per centimeter of caliper width is sufficient to accomplish effective gene transfer.
Analysis of muscle fiber cross-sectional area and whole muscle contractility after electroporation are important aspects of the method for measuring changes in muscle size and function due to gene modulation. We and others have previously demonstrated that electroporation of control vectors alone does not cause a decrease in myofiber area. The green fluorescent protein (EGFP) construct was a useful fluorescent indicator of DNA transfection in these studies13,14. A number of studies have investigated in situ contractility of the TA after electroporation and found varying results. One study showed that 75 V/cm electroporation caused about a 30% reduction in tetanic force 3 days post-electroporation, and by 7 days post-electroporation, tetanic force was back to the control level, while 50 V/cm electroporation did not compromise force13,15. Another study showed that there was a 30% loss of tetanic force 3 h after 180 V/cm electroporation, which recovered to the sham force levels after 7 days16.
In the following detailed procedure, we demonstrate injection and electroporation of a pcDNA3-EGFP plasmid in the TA and extensor digitorum longus (EDL) muscles of mice. We also demonstrate that this method does not affect EDL whole-muscle contractility. The aim is to demonstrate efficient plasmid uptake into myofibers without causing loss of function.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-0 Nylon suture (non-absorbable)</td>
			<td>Ethicon</td>
			<td>662G</td>
			<td>Suture to close skin incision</td>
		</tr>
		<tr>
			<td>50&#181;l Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80501</td>
			<td>microsyringe</td>
		</tr>
		<tr>
			<td>C57BLl/6NHsd mice</td>
			<td>Envigo</td>
			<td>044</td>
			<td>12 week-old female mice used for experimentation</td>
		</tr>
		<tr>
			<td>Caliper Electrode</td>
			<td>BTX</td>
			<td>45-0102</td>
			<td>1.0cm x 1.0cm stainless steel</td>
		</tr>
		<tr>
			<td>Dynamic Muscle Control Data Acquisition/analysis</td>
			<td>Aurora Scientific</td>
			<td>605A</td>
			<td>Software used for muscle contractility measurement and analysis</td>
		</tr>
		<tr>
			<td>ECM 830 Electroporation System</td>
			<td>BTX</td>
			<td>45-0662</td>
			<td>electroporator</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>Plasmid purification kit</td>
		</tr>
		<tr>
			<td>Extra Narrow Scissors</td>
			<td>Fine Science Tools</td>
			<td>14088-10</td>
			<td>Scissors for blunt dissection</td>
		</tr>
		<tr>
			<td>Force Transducer</td>
			<td>Aurora Scientific</td>
			<td>407A</td>
			<td>To measure force from EDL</td>
		</tr>
		<tr>
			<td>Micro-Masquito Hemastats</td>
			<td>Fine Science Tools</td>
			<td>13010-12</td>
			<td>Hemastats for surturing</td>
		</tr>
		<tr>
			<td>pcDNA3.1 mammalian expression vector</td>
			<td>Fisher Scientific</td>
			<td>V79020</td>
			<td>Control Vector</td>
		</tr>
		<tr>
			<td>pcDNA3-EGFP expression plasmid</td>
			<td>Addgene</td>
			<td>13031</td>
			<td>Plasmid for GFP expression</td>
		</tr>
		<tr>
			<td>Semken curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11009-13</td>
			<td>Forceps for surgery</td>
		</tr>
		<tr>
			<td>Surgical blades stainless steel no. 10</td>
			<td>Becton Dickinson</td>
			<td>37 1210</td>
			<td>Scalpel blades</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. media</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Freezing media for histology</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin- Texas Red</td>
			<td>Thermo-Fisher Scientific</td>
			<td>W21405</td>
			<td>Membrane staining for muscle cross section</td>
		</tr>
	</tbody>
</table>

electroporation of plasmid dna into mouse skeletal muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation to facilitate gene transfer in skeletal muscle is a useful technique used to evaluate changes in muscle physiology. We have demonstrated a detailed, step-by-step procedure to accomplish efficient gene transfer in both the TA and EDL muscles. Differences in transfection efficiency occur due to a number of variables. Among these variables are electroporation parameters (pulses, voltage, pulse duration, etc.), gene construct size, and concentration/volume of DNA injected. We have previously shown that electr...

Watch this Scientific Journal Video about Electroporation of Plasmid DNA into Mouse Skeletal Muscle at JoVE.com

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

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