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High efficiency transfection of embryonic limb mesenchyme with plasmid DNA using square wave pulse electroporation and sucrose buffer
Brent E. Bobick1, Peter G. Alexander1,2, and Rocky S. Tuan1,2
1Department of Health and Human Services, Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD
2Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, PA

Present address: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
BioTechniques, Vol. 56, No. 2, February 2014, pp. 85–89
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Micromass cultures of primary embryonic limb mesenchyme are a valuable model system for studying cartilage formation in vitro. However, high efficiency introduction of plasmid DNA into this hard-to-transfect cell type typically results in considerable cell death and significantly impeded chondrogenesis when the cells are subsequently plated in high density micromass. Here, we describe a novel method in which square wave pulse electroporation of chick embryo wing bud mesenchyme suspended in protective sucrose buffer results in high efficiency transfection without substantially affecting micromass culture cell viability or chondrogenic differentiation potential. Furthermore, we show that this protocol can be employed, along with effector gene expression vectors, to generate observable changes in the amount of cartilage tissue formed in micromass, unlike lower efficiency, higher cytotoxicity techniques that require co-transfection of reporter plasmids to monitor changes in the extent of chondrogenesis and correct for differences in cell viability.

High density, three dimensional micromass cultures of prechondrogenic limb mesenchyme have been widely employed for in vitro recapitulation of embryonic cartilage formation (i.e., chondrogenesis) since they were first proposed as a model of this developmental program by Solursh and colleagues over 35 years ago (1). Experiments conducted in micromass cultures of dissociated limb mesenchymal tissue have contributed extensively to our understanding of normal developmental chondrogenesis. Importantly, the sequence of cellular and molecular events that occur during cartilage formation in the developing limb is mimicked in micromass culture. Both in vivo and in micromass, mesenchymal progenitor cells undergo condensation into compact aggregates prior to up-regulating expression of the chondrogenic master gene Sox9, differentiating into chondrocytes, and synthesizing cartilage-characteristic extracellular matrix (ECM) molecules such as collagen type II and the proteoglycan aggrecan (reviewed in Reference (2). Although micromass cultures are amenable to the addition of exogenous growth factors, pharmacological inhibitors, antibodies, and other molecules, our ability to introduce plasmid DNA into the hard-to-transfect primary limb bud mesenchymal cells that often comprise these cultures is less than adequate (3-5). Moreover, because high chondroprogenitor cell density is a requirement for chondrogenesis both in vivo and in vitro (reviewed in 2), the customary methodological trade-off in which decreased cell viability is exchanged for increased transfection efficiency results in a distinct lack of cellular differentiation in this model system.

Method summary

We describe a new method for transfection of plasmid DNA into hard-to-transfect primary chick embryo wing bud mesenchymal cells that are subsequently cultured in micromass and differentiated into chondrocytes. Our technique, which couples square wave pulse electroporation and a protective sucrose buffer, is both highly efficient and minimally cytotoxic. Thus, cells transfected with this electroporation protocol maintain a high density in culture and undergo normal levels of micromass cartilage formation. This obviates the need for reporter plasmids to monitor the extent of differentiation and correct for differences in cell viability.

Previous studies by our group have evaluated a range of plasmid DNA transfection techniques for their effectiveness with suspensions of dissociated chick embryo wing bud mesenchyme. Both exponential decay electroporation using the presently discontinued Electroporator II from Invitrogen and molecular vibration-mediated transfection with the Gene Symphonizer (Mollennium Laboratories) achieved high efficiency, ~35% and ~78% respectively, but left only one-third of chick wing bud mesenchyme viable (3, 4). When commonly used, commercially available transfection reagents like FuGENE 6 (Promega) are optimized to be gentle on limb mesenchyme, the transfection efficiency plummets to a few percent (4). A more recent report (5) found that the transfection reagents SuperFect (Qiagen), Lipofectamine 2000, and DMRIE-C (both from Life Technologies) can deliver plasmid DNA in a manner that is only slightly detrimental to wing bud mesenchyme, but this results in transfection efficiencies that peak at ~25% of the cells. Amaxa nucleofection (Lonza) and the transfection reagent SAINT-MIX (Synvolux Therapeutics) achieved much higher efficiencies, ~97% and ~55% respectively; however, both of these methods simultaneously reduced proliferation and increased the presence of early apoptotic cells (5). Viral transduction of plasmid DNA into dissociated embryonic chick limb mesenchyme is characterized by high efficiency and low cytotoxicity (6). Unfortunately, the time required for retroviral integration renders transduction less useful for studying the early events of chondrogenesis in this model as cellular condensation and the beginnings of overt chondrocyte differentiation occur in the first 24 h of micromass culture (reviewed in 7).

A new method recently developed by Underhill and colleagues employs the reagent Effectene (Qiagen) and buffer supplemented with 400 mM trehalose to transfect plasmid DNA into dissociated mouse embryo limb bud mesenchymal cells in suspension (8, 9). Interestingly, organisms that are tolerant of extreme environmental stress, such as desiccation, accumulate large amounts of disaccharides, most often trehalose or sucrose, in the dehydrated state. These disaccharides are known to directly interact with lipids and have been shown to stabilize biological membranes (reviewed in References (10, and 11). We hypothesized that electroporation buffer containing sucrose might protect wing bud mesenchyme from the membrane insult of electroporation and increase cell viability following high voltage pulsing. In this study, we describe a novel transfection protocol that employs square wave pulse electroporation and buffered sucrose as an electroporation solution to introduce plasmid DNA into suspensions of primary chick embryo wing bud mesenchymal cells with both high efficiency and limited cytotoxicity. Moreover, we show the power of this new technique to introduce both expression and reporter vectors into prechondrogenic mesenchyme that can subsequently be plated in micromass and differentiated into chondrocytes.

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