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Various methods have been developed to introduce the exogenous gene into primary neurons, and neuroblastoma, such as electroporation (nuclear transfection), calcium phosphate, viral vectors, and magnetic and physical transfection. Each approach has its advantages and limitations.
Ca2+-phosphate/DNA coprecipitation method is one of the most mature transfection methods, which is very commonly used in vitro transfection of different types of neuron cells and cell lines. It is cost-effective, requires no specialized equipment, and is easy to set up. The method can be used to transfect neurons at all stages of differentiation, including those that have already formed functional neural networks.
An advantage of this method is that titrating DNA concentration by changing the amount of plasmids used makes it easy to change the time course and level of protein expression. This is an advantage because rapid and intense expression reduces the time that an overexpressed protein appears at (near) physiological levels before it can cause an overexpressed artifact. Importantly, after optimization, transfection with Ca2+-phosphate/DNA coprecipitation resulted in good cell viability. These advantages make this method ideal for applications where a small number of transfected cells need to display physiologically normal behavior. These include, for example, in vivo imaging experiments that focus on in vitro single cells found in a network of cultured neurons (a low number of transfected cells in a complex network of neurons is an advantage when dendrites and axons of a single neuron must be identified) or assessing neuronal phenotypes after RNAi. The method can also be used to study the subcellular localization of proteins and the co-localization of proteins and RNA in developing and mature neurons.
Lipofection is considered the "gold standard" to which other techniques are usually benchmarked due to its ability to efficiently introduce nucleic acids (DNA and RNAi) into a broad range of cell types. Conventional lipid-mediated gene delivery is based on cationic lipid molecules that form small unilamellar that interact with negatively charged nucleic acids (NAs) and promote the fusion of lipids: NA complexes with negatively charged plasma membranes. Cationic lipid molecules typically bind to neutral helper lipids that mediate the fusion of liposomes with membranes However, newer generation liposomal transfection reagents use non-liposomal lipids to form complexes with NA that are thought to be endocytosed and released into the cytoplasm. Lipofection techniques are simple, require no specialized equipment, exhibit high reproducibility and low toxicity, and generally require little optimization (although several reagents may need to be tested to get the best results on unconventional cell lines/types) they are suitable for instantaneous and stable transfection of multiple cell lines.
This is a novel transfection technique based on DNA-coated magnetic nanobead delivery, which can be used to transfect neurons and neuroblasts. Particle complexes consisting of iron oxide and nucleic acid are formed through salt-induced colloid aggregation and electrostatic interactions. This rapid, non-viral program may be used only in adherent cells, but it is a common technology adapter for many types of nucleic acids. After the nucleic acid-particle complexes are added to the cell, they are transported directly to the magnetic plate as a magnetic field source. It can improve the efficiency of the process, even if a small amount of DNA is applied. Nucleic acids are suitable for uptake through endocytosis and pinocytosis, thus keeping the membrane structure intact.
During microinjection, the nucleic acid is injected into the cytoplasm or nucleus using a fine glass capillary. While microinjection has been used in mammalian neurons and neuroblasts. It is more commonly used in experiments with (larger and more robust) invertebrate neurons. A major disadvantage of this technique is the tremendous pressure caused by the destruction of the plasma membrane during microinjection, which leads to very low survival rates for many types of neurons. Importantly, to ensure that the injection does not impair the integrity and function of the neurons and/or subsequent development, each microinjection experiment must contain appropriate controls. Despite its drawbacks, this technique has major advantages: it allows the injection of substances that cells cannot synthesize. For example, directly labeled RNAs (including micro-RNAs) can be applied to track their subcellular localization and turnover or their association with specific proteins or other RNAs.
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