Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 conditions^{1,2,3,4,5,6,7,8,9}. 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 studies^13,14. 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 force^13,15. 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.

Protokół

All experiments using animals were performed at the Penn State College of Medicine, approved by Penn State University'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

NOTE: These steps are identical for TA and EDL injection/electroporation preparation.

1. Purify expression plasmid constructs to a concentration of 1 µg/µL diluted in sterile PBS prior to the experiment. For this procedure, a commercially available endotoxin-free purification kit was used to purify pcDNA3-EGFP (GFP) or pcDNA3 empty vector (control).
2. Calculate the plasmid concentration to ensure adequate injection volume (TA 50 µg of DNA in 50 µL of sterile PBS; EDL 10 µg of DNA in 10 µL of sterile PBS) and aliquot samples.
3. Program the electroporator settings by using the select wheel and depressing the wheel to choose the parameters as follows: Mode - LV, Voltage - 12.5 V/mm (to be adjusted at the time of injection), pulse length - 20 ms, number of pulses - 5, interval - 200 ms, polarity - unipolar.
4. Anesthetize the mice using isoflurane gas. Place the mice in an induction box with 5% isoflurane. Confirm the surgical plane of anesthesia by the absence of the toe pinch reflex using forceps and reduce isoflurane to 2% maintenance dose. Transfer mice to an appropriate nose cone resting on a circulating water plate at 37 °C for the rest of the procedure.
5. Remove hair from both hindlimbs using small hair clippers. Scrub the lower limbs with alternating 70% ethanol/betadine to sanitize the injection area.

2. TA injection/electroporation

1. With the mouse in a supine position, locate the TA tendon visible through the skin on the lateral side of the lower leg. Using a 50 µL micro-syringe with a detachable 30 G needle, insert the needle 1-2 mm superior to the myotendinous junction at a shallow 5° angle until the needle reaches the superior end of the muscle.
   NOTE: Injection into the middle of the muscle is the goal.
2. Slowly depress the plunger while slowly retracting the needle along the injection path to deliver 50 µL of plasmid solution. The muscle should swell.
3. Set the timer for 1 min and measure the thickness of the leg at the TA. Depending on the size of the mouse, this can be from 5-10 mm. Set the caliper electrodes to the measured thickness and set the electroporator voltage to 12.5 V/mm.
4. After 1 min, place the caliper electrodes around the lower limb. The electrodes should be snug but not overly tight. Deliver 5 square-wave pulses, with 20 ms duration and 200 ms intervals (the muscle should twitch with each pulse).
5. Repeat with the alternate limb using the control vector.
6. Remove the mouse from anesthesia and allow it to recover on a heating pad set to 37 °C. Once recovered, return the mouse to its cage.

3. EDL injection/electroporation

1. With the mouse in a supine position, locate the tibia bone anterior crest visually and through gentle palpation.
   1. Using a scalpel, make a shallow incision through the skin on the lateral side of the tibia anterior crest 5 mm inferior to the knee to 2 mm superior to the TA myotendinous junction.
   2. Using small scissors, blunt dissect the fascia, exposing the TA muscle.
   3. Again, using blunt dissection with scissors, separate the TA muscle from the tibia gently by pulling the muscle laterally, exposing the EDL. Small, curved forceps can be used to keep the TA clear from the EDL during the procedure.
2. Using a 50 µL micro-syringe with a detachable 30 G needle, insert the needle into the EDL longitudinally until the needle reaches the superior end of the muscle.
3. Slowly depress the plunger while slowly retracting the needle along the injection path to inject 10 µL of the plasmid solution (muscle should swell).
4. Set a timer for 1 min and measure the thickness of the leg at the EDL. Depending on the size of the mouse, this can be from 5-10 mm. Set the caliper electrodes to the measured thickness and set the electroporator voltage to 12.5 V/mm.
5. After 1 min, place the caliper electrodes around the lower limb. The electrodes should be snug but not overly tight. Deliver 5 square-wave pulses, with 20 ms duration and 200 ms intervals (muscle should twitch with each pulse).
6. Close the incision using disposable 4/0 non-absorbable nylon sutures.
7. Repeat with the alternate limb or leave the limb untouched as absolute control.
8. Remove the mouse from anesthesia and allow it to recover on a heating pad set to 37 °C. Administer an appropriate subcutaneous analgesic immediately after surgery and 12-24 h following surgery. Once recovered, return the mouse to its cage.
   NOTE: Previous studies have shown a muscle contractility deficit in the TA immediately after electroporation and that contractile recovery occurs over the course of 3 days^13,15. For this reason, the analysis of both the TA and EDL muscles for histology, protein expression, or muscle contractility is conducted 3-10 days following electroporation. Prolonged expression of EGFP has been observed up to 3 weeks following the procedure¹³.

Wyniki

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...

Dyskusje

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...

Materiały

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