DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

Summary

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Abstract

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver¹, skeletal muscle^2,3, and even the brain⁴. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP³, calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca²⁺ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical³ and functional evidence⁵. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions^2,6-8.

Protocol

Experimental Procedures for in vivo electroporation of FDB and IO muscles

1. Before starting the in vivo electroporation protocols, mammalian expression plasmids must be amplified to yield concentration in the range of to 2-5 μg plasmid/μl of TE. Note: We routinely use commercial amplification kits and follow the manufacturer’ procedures. Commercial expression plasmids carrying the CMV promoter work very well in skeletal muscle in vivo transfections.
2. Aliquot the necessary volume (10-20 μl) of the plasmid solution and save it in two 0.5 ml Eppendorf tubes (one for each foot of the mouse).
3. Prepare a solution containi....

Discussion

We describe here the detailed steps that should be followed in order to attain effective transfections of DNA plasmids into skeletal muscle fibers by in vivo electroporation. The main advantages of our approach are the simplicity of implementation, and its minimal invasiveness which results in negligible health hazard to the animals. In reality, the above described electroporation protocols do not entail much more than two subcutaneous injections per foot, followed by an electrical stimulation protocol, all of w.......