Transfecting and Transducing Neurons with Synthetic Nucleic Acids and Biologically Active Macromolecules

Miro1 Is a Calcium Sensor for Glutamate Receptor-Dependent Localization of Mitochondria at Synapses

“…To determine whether the increase in mitochondrial motility that we observed upon Miro1 expression was dependent on enhanced coupling to KIF5, we introduced into neurons transfected with Miro1 a KIF5 function-blocking antibody (SUK4; Ingold et al., 1988; Jaulin et al., 2007 ) that does not inhibit myosin- or dynein-based motility ( Bi et al., 1997; Lane and Allan, 1999 ), or a control antibody (9E10). This was done by complexing the antibody with a membrane-permeant carrier peptide ( Figure S5 ; Kittler et al., 2006; Morris et al., 2001 ). The KIF5 function-blocking antibody strikingly lowered the fraction of moving mitochondria in neurons transfected with Miro1 ( Figures 1 H–1J: 5.9% ± 1.8% of mitochondria were moving in Miro1-transfected neurons treated with SUK4 antibody, compared to 42.0% ± 5.2% in Miro1-transfected neurons treated with control 9E10 antibody, p = 6.8 × 10 −6 ), confirming that Miro1-dependent facilitation of mitochondrial transport is due to Miro1 increasing the fraction of mitochondria that are coupled to KIF5 motor proteins.…”

“…For all experiments, except where stated otherwise, neurons were transfected at DIV 6–8 using a calcium phosphate method as described ( Xia et al., 1996 ). To quantify RNAi knockdown of Miro1, shRNAi constructs were transfected using Amaxa nucleofection, which has a transfection efficiency of 50%–60% ( Kittler et al., 2004, 2006 ). For experiments coculturing two populations of neurons transfected with either mtdsred2 or GFP-synaptophysin, transfection was by Amaxa nucleofection, and the two sets of cells were mixed before plating.…”

“…For experiments coculturing two populations of neurons transfected with either mtdsred2 or GFP-synaptophysin, transfection was by Amaxa nucleofection, and the two sets of cells were mixed before plating. For antibody transduction of neurons, affinity-purified SUK4 or 9E10 antibody as a control were transduced across the plasma membrane at DIV 10–14 using Chariot reagent (Active Motif) following the manufacturer's instructions with minor modifications ( Kittler et al., 2006; Morris et al., 2001; Payne et al., 2003; Remacle et al., 2005; Sloane and Vartanian, 2007 ). In brief, a 100 μl mix of 2 μl of Chariot reagent complexed with 10 μg of antibody was overlaid onto cultured neurons with 100 μl serum-free media and incubated at 37°C.…”

Delivery of GABAARs to Synapses Is Mediated by HAP1-KIF5 and Disrupted by Mutant Huntingtin

“…+/+ and 109Q/109Q cultures for live video microscopy were transfected using Amaxa nucleofector technology using programme T27 and plated onto coverslips for imaging. Cultures for surface labelling and analysis of GABA A R clusters were transfected by a calcium phosphate method as previously described (Kittler et al, 2006) at 7–10 DIV and allowed to express for at least three days before fixation. Monoclonal antibodies (affinity purified SUK4, or 9E10 antibody as a control) were transduced across the plasma membrane of DIV 10–14 neurons using Chariot reagent (Active Motif) following the manufacturer’s instructions with minor modifications (Coulpier et al, 2002; Kittler et al, 2006; Ma et al, 2006).…”

From Cultured Rodent Neurons to Human Brain Tissue: Model Systems for Pharmacological and Translational Neuroscience

“…In order to generate the right model system, the manipulation of neural cell cultures` intrinsic properties is often necessary. Through the use of transfections, infections, electroporation, and microinjections 18 , as well as de-novo mutations in transgenic mouse lines 19 , the introduction of genetic elements such as coding sequences, shRNA, and more can be controlled. This facilitates the generation of cell cultures that express channels, receptors, or any specific gene of interest, either as mutated forms or their wild-type counterparts, for functional binding, signaling, and electrophysiological studies.…”