DNA Transfection of Mammalian Skeletal Muscles using <em>In Vivo</em> Electroporation

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 conditions2,6–8.

Skeletal muscle-specific T-tubule protein STAC3 mediates voltage-induced Ca 2+ release and contractility

“…As a first step toward understanding the function of STAC3, we determined the subcellular localization of the protein by electroporating an expression plasmid encoding a fusion protein of STAC3-GFP (GFP at the carboxyl end) into mouse flexor digitorum brevis muscles (FDB) in vivo. Following a 3-d recovery period, the FDB was imaged directly using two-photon laser scanning microscopy with second harmonic generation to visualize the myosin A bands as an internal reference, as described by DiFranco et al (14). STAC3-GFP localized in a repeating doublet pattern with the wider spaces correlating to the myosin heavy chains (Fig.…”

“…Flexor digitorum brevis muscles were electroporated following a published protocol (14). Following a 3-d recovery, the mice were killed, their feet were skinned, and the unfixed FDB was examined directly in imaging buffer by two-photon laser scanning microscopy (Zeiss; LSM 780) with reverse second harmonic generation to visualize the A bands as an internal reference.…”

A Novel MitoTimer Reporter Gene for Mitochondrial Content, Structure, Stress, and Damage in Vivo

“…All experimental protocols were approved by the University of Virginia, Institutional Animal Care and Use Committee. pMitoTimer and pLamp1-YFP transfection by somatic gene transfer in the adult mouse FDB muscle was performed as previously published (30). Briefly, the mice were anesthetized (isofluorane) and the base of each hind limb foot, where the flexor digitorum brevis (FDB) muscle is located, was injected with 10 l of hyaluronidase (0.36 mg/ml; Sigma) subcutaneously.…”

Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair

“…Flexor digitorum brevis (FDB) fibers were transfected by in vivo electroporation methods described in detail in ref. 46. Muscle fibers were isolated as above and studied 7 d after electroporation to allow for recovery and protein expression in the electroporated muscles.…”