doi:10.1016/j.pep.2005.10.018
Copyright © 2005 Elsevier Inc. All rights reserved.
Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system
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Marino DiFrancoa, Patricia Ñecoa, Joana Capotea, Pratap Meerab and Julio L. Vergaraa, 
, 
aDepartment of Physiology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA
bDepartment of Neurobiology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA
Received 2 September 2005.
Available online 11 November 2005.
Abstract
The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of 
1 mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.
Keywords: Transgenic protein expression; In vivo electroporation; Fluorescent proteins; Gene expression; Mammalian skeletal muscle
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Fig. 1. Optical assessment of transfection efficiency of pEGFP-N2 in skeletal muscle. (A1) Color image of an FDB muscle dissected 5 days after transfection with pEGFP when illuminated with white light. (A2) Image from the same muscle when illuminated with monochromatic blue light (480 nm) and the fluorescence filtered with a 550 nm long pass filter. Both images were obtained with a 4 Mega pixels digital camera attached to a dissecting microscope. (B1) Image of an FDB muscle dissected 12 h after transfection. Image was obtained by stacking 11 consecutive TPLSCM sections. The image was rendered in 256 intensity levels of green, spanning a fluorescence scale of 0–1500 arbitrary units (AU) in the TPLSCM. (B2) Same as above, but for an FDB muscle dissected 5 days after transfection. The 256 intensity levels of green span a fluorescence scale of 0–65,536 AU. For both (B1) and (B2), the microscope objective was an Olympus 10×, NA 0.25 and the calibration bars represent 200 μm. (C) Single TPLSCM section image through a bundle of fibers from the same muscle shown in (B2). Muscle was slightly stretched. The inset in (C) represents an intensity profile measured along the areas indicated by the orange rectangles. Notice the breaks in the ordinate axis. For the image shown in (C), the microscope objective was an Olympus 20×, NA 0.95 (Olympus XLUMPLANFL) and the length of the orange rectangle is 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
Fig. 2. Biochemical characterization of EGFP expressed in FDB muscles. (A) SDS–PAGE of supernatant fractions obtained from a control (lane 1) and a transfected (lane 2) FDB muscle. Lane MWM contains the molecular weight markers. The arrow indicates a band corresponding to an apparent molecular weight of 26.6 kDa. Each lane was loaded with 7.29 μg total protein. (B) Western Blot analysis of a replica of the SDS–PAGE shown in (A). (C) Fluorescence emission spectra of 1:20 dilution of the supernatant obtained from a pEGFP-transfected FDB muscle (trace a), a control muscle (trace c), and 10 μg/ml commercial EGFP (trace b). The wet weight of the transfected muscle was 11.9 mg and the total supernatant volume was 75.8 μl. (D) Traces a and b of (C) were normalized to their respective peaks at 508 nm and shown superimposed.
Fig. 3. Time course of expression of EGFP in lower limb muscles. (A) SDS–PAGE of supernatant fractions obtained from lower limb muscles transfected with pEGFP. Samples were taken after 0.5, 1, 2, 4, 8, 16, 24, and 31 days from transfection for lanes 1–8, respectively. The lane containing the molecular weight markers is labeled as MWM. The arrowhead indicates the position of a band corresponding to an apparent molecular weight of 26.7 kDa. Each lane was loaded with 10 μg total protein. (B) Western blot of a replica of the gel shown in (A).
Fig. 4. Time course of EGFP expression yield in lower limb muscle. Bar graph of the EGFP yield, expressed in milligram of EGFP per gram wet weight of lower limb muscle tissue, plotted as a function of the time after muscle electroporation. Both axes are displayed in logarithmic scales. Experimental data were obtained in duplicates for each time point and drawn superimposed in the graph.
Table 1.
Levels of protein extracted from supernatant fractions of lower limb muscles at different periods after transfection
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