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Abstract
The development of gene transfection technologies has greatly advanced our understanding of life sciences. While use of viral vectors has clear efficacy, it requires specific expertise and biological containment conditions. Electroporation has become an effective and commonly used method for introducing DNA into neurons and in intact brain tissue. The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5–16. This cell type has been particularly recalcitrant and refractory to physical or chemical methods for introduction of DNA. By optimizing the culture condition and parameters including voltage and duration for this specific electroporation system, high efficiency (60–80%) and low toxicity (>60% survival) were achieved with robust differentiation in response to Nerve growth factor (NGF). Moreover, 3–50 times fewer cells are needed (6 × 104) compared with other traditional electroporation methods. This approach underlines the efficacy of this type of electroporation, particularly when only limited amount of cells can be obtained, and is expected to greatly facilitate the study of gene function in dorsal root ganglia neuron cultures.
Highlights
Culture of primary cells has been extensively used to study neuronal survival, signal transduction, development, and neurite outgrowth
The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5–16
Dorsal root ganglia (DRG)-derived sensory neurons that are selectively sensitive to Nerve growth factor (NGF), Brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT3), provide an excellent model in which to study the mechanisms of axonal regeneration, neurotrophin signaling, peripheral nervous system development and peripheral neuron disease (Melli and Hoke, 2009; Newbern et al, 2011)
Summary
Culture of primary cells has been extensively used to study neuronal survival, signal transduction, development, and neurite outgrowth Gene transfer, through both viral and non-viral methods, has become a powerful technique to assess the effects of expression of selected genes. The Amaxa Nucleofector system, one of the best known and commonly performed transfection methods in the labs, requires 1 × 106 DRG cells in 100 μl for each electroporation [e.g., Chick DRG (Chadborn et al, 2006); Manufacturer’s instructions]. This is a major obstacle when working with embryos. Though encouraging results have been reported recently with adult Rat DRG using the 4D-Nucleofector system-X from Lonza (McCall et al, 2012), electroporation of dissociated DRG neurons, from young mouse embryos, whose cell number is limiting, remains a challenge
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