CORP: Gene delivery into murine skeletal muscle using in vivo electroporation

Abstract

The strategy of gene delivery into skeletal muscles has provided exciting avenues in identifying new potential therapeutics toward muscular disorders and addressing basic research questions in muscle physiology through overexpression and knockdown studies. In vivo electroporation methodology offers a simple, rapidly effective technique for the delivery of plasmid DNA into postmitotic skeletal muscle fibers and the ability to easily explore the molecular mechanisms of skeletal muscle plasticity. The purpose of this review is to describe how to robustly electroporate plasmid DNA into different hindlimb muscles of rodent models. Furthermore, key parameters (e.g., voltage, hyaluronidase, and plasmid concentration) that contribute to the successful introduction of plasmid DNA into skeletal muscle fibers will be discussed. In addition, details on processing tissue for immunohistochemistry and fiber cross-sectional area (CSA) analysis will be outlined. The overall goal of this review is to provide the basic and necessary information needed for successful implementation of in vivo electroporation of plasmid DNA and thus open new avenues of discovery research in skeletal muscle physiology.

Keywords: atrophy; electroporation; gene transfer; hypertrophy; protein synthesis.

Graphical abstract

Figure 1.

Proposed mechanisms for the movement of plasmid DNA into electroporated muscle fibers. Generally, in vivo electroporation experiments allow for a within-animal study design to be used where one side of the animal is electroporated with an expression plasmid containing a gene of interest and the contralateral leg serves as the empty vector control. A: after plasmid injection, each leg is exposed to electrical pulses through an electroporation generator via electrodes placed on the targeted hindlimb muscle. B: application of electrical pulses to the muscle generates positive and negative charge on either side of the membrane. In turn, this charge imbalance leads to the formation of hydrophobic pore defects enabling plasmid DNA to enter the myofiber. C: movement of plasmid DNA into the myofiber is proposed to occur either through movement toward the anode electrophoretically or by localizing with other DNA molecules at the cell membrane and moving together into the myofiber as a complex without electrophoretic forces being required. D: these proposed mechanisms contribute to the successful introduction of the plasmid DNA into the cell resulting in a transfected myofiber [depicted as green fluorescent protein (GFP) positive]. Adapted from McMahon and Wells (27). Created with Biorender.com.

Figure 2.

Applications of plasmid DNA into skeletal muscle physiology research. A: use of mammalian expression plasmids can aid in mechanistic experiments because they can be introduced by transfection (e.g., electroporation) into host cells. Expression plasmids possess a multiple cloning site (MCS) immediately downstream of a promoter sequence that allows for the amplified cDNA of a gene of interest to be cloned into the plasmid. The MCS of a plasmid consists of numerous unique restriction enzyme recognition sequences that simplify the cloning process and allow for the proper directional insertion of the cDNA of a gene of interest. Furthermore, the use of a viral promotor (e.g., CMV or SV40) allows for robust expression of the target gene within eukaryotic cells. Finally, the cDNA cloned into an expression plasmid can be easily sequenced using commonly used primer sites (e.g., T7, SP6, and T3) to confirm successful target gene insertion and to verify that no mutations have been introduced during the cloning process. B: transcriptional regulation of a gene in skeletal muscle under various stimuli can be ascertained by using a reporter plasmid construct containing the promoter of a gene of interest inserted upstream of a reporter gene (e.g., luciferase). For example, in immobilized/disuse muscle atrophy, the target gene promoter activity can be measured in control and immobilized tissues that have been transfected with the reporter plasmid construct. Under an atrophy stimulus, the gene of interest promoter activity will be detected through changes in the amount of luciferase activity. C: use of a fluorescent reporter, such as green fluorescent protein (GFP), allows for the identification of successfully transfected muscle fibers under the microscope. The incorporation of a GFP-tag into a target protein can aid in studying the localization of novel proteins in skeletal muscle fibers. D: to investigate protein-protein interactions and complexes, epitope tags [e.g., hemagglutinin (HA), FLAG, myc, and glutathione S-transferase (GST)] provide a useful tool that can be used in common molecular biology techniques such as coimmunoprecipitation to identify novel interactors of the protein of interest. Most commonly, the epitope tag is integrated into the plasmid sequence so that the tag will be easily added in frame with either the C- or N-terminus of the target protein. In addition, these tags can be used to identify proteins when a specific antibody against a protein of interest is not yet available. Created with Biorender.com.

Figure 3.

Injection site, number, and electrode placement in mouse hindlimb muscles for in vivo electroporation. In mouse quadriceps, gastrocnemius (GA) and tibialis anterior (TA) muscles, single injections can be applied using 0.5-mL insulin syringe (28-gauge) to the distal area of the muscles, as depicted by the white X mark (A). In the GA muscle, the lateral and medial GA muscle require an injection in each head of the muscle. Additional injections can be applied to the TA muscle at the proximal end and mid belly of the muscle (depicted as black X mark) and this can be beneficial if utilized within the rat TA muscle. In B, electrode placement is illustrated for the GA and TA muscles from an overhead and side view. For the GA muscles (Bi), plate electrodes (∼10 mm size) are placed on each side of the leg, one plate covering the lateral and the other plate covering medial GA muscle area. For the TA muscle (Bii), electrode plates (∼7 mm size) are placed, with one plate on top of the mid belly of the muscle between the distal and proximal end. The other plate is located at the back of the leg over the GA muscles. Good contact between the electrode and skin is enhanced with removal of the hair and use of a conductive gel. Created with Biorender.com.

Figure 4.

Identification and quantification of transfected skeletal muscle fibers in various hindlimb muscles. A: representative images of hindlimb skeletal muscles from 12- to 16-wk-old C57/BL6 male mice electroporated with an emerald GFP (emGFP) plasmid for 7 days. Separately, tibialis anterior (TA) and gastrocnemius (GA) muscles are given an intramuscular injection with 0.4 U/μL of hyaluronidase before electroporation. After 2 h, the TA and GA muscles are then injected with 2 µg and 5 µg of emGFP respectively and electrical pulses (175 V/cm, 10 pulses, 20 ms) are applied using two-paddle plate electrodes as detailed in published protocols (54, 67, 84). The TA and gastrocnemius muscles were stained for hematoxylin and eosin (H&E), and laminin using protocols detailed in Table 5. ×10 magnification, scale bar = 100 µm. In the analysis of transfected muscle fiber cross-sectional area (CSA), comparisons can be made between empty vector and gene of interest transfected fibers in unchallenged skeletal muscle to assess for hypertrophy (B; yellow line) and atrophy (C; black line). The blue line represents normal fiber CSA distribution of nontransfected or empty vector fibers in B and C. The nontransfected fibers can be assessed for fiber CSA to confirm that the plasmids are not impacting surrounding local muscle fibers. D: in transfected muscle fibers undergoing an additional intervention (e.g., denervation), fiber CSA comparisons between empty vector (black line), gene of interest (purple line), and nontransfected/innervated (blue line) fibers can be analyzed. E: an alternative strategy to incorporate nontransfected muscle fibers in the CSA quantification is to represent the fiber CSA as a percentage of the nontransfected fibers and compare the empty vector to the gene of interest expression plasmid, as used in previously published studies (57,58, 157). Created with Biorender.com.