Transfection Techniques for Neuronal Cells

Summary of advantages and disadvantages of different techniques commonly used to transfect mammalian neurons

	Best suited for	Strengths	Limitations	Toxicity	Onset, level, and duration of expression^{^a}	Maximum insert size	Genome integration
Electroporation	Cell types/tissues: Neuronal cell lines and freshly isolated primary neuronal cells in vitro; whole embryos in vivo Applications: Transfection of large numbers of robust cells in vitro (in suspension) or in vivo when high transfection efficiencies are required	Simple and quick protocol, relatively little optimization required	Can only be used for freshly isolated neurons or cells in suspension that have yet to produce neurites; transfection efficiencies limited by premature voltage pulse termination; relatively expensive equipment and reagents	Variable depending on cell type and electroporation parameters used (robust cell types tend to survive the procedure better)	Typically within hours; expression levels variable depending on electroporation parameters used	No limit	No
Nucleofection	Cell types: Neuronal cell lines and freshly isolated primary neuronal cells in vitro Applications: Quantitative and biochemical analyses because of very high transfection efficiencies, e.g., assessment of protein down-regulation after RNAi-mediated knock-down; introduction of genetic material into neural progenitor cells with subsequent expansion and/or differentiation	Very high transfection efficiencies (typically ∼50%; up to 95% after optimization); often results in nuclear localization of transfected plasmids yielding higher expression rates; reproducible and simple to perform once the procedure has been optimized	Can only be used for freshly isolated neurons or cells in suspension that have yet to produce neurites; relatively expensive equipment and reagents; can require optimization of programs and nucleofection solutions used	Relatively low cell toxicity because of finely regulated sequences of voltage pulses and cell type-specific nucleofection solutions	Typically within hours; moderate expression rates, therefore possible to harvest cells up to several days after transfection to assess, for example, maximal downregulation of target proteins or long-term phenotypes	No limit	No
Single cell electroporation	Cell types: Individual neuronal cells in vitro or in vivo, including mature, fully differentiated neurons Applications: Transfection of neurons in brain slices and in intact brains of living animals; assessment of the morphology, function, and behavior of single cells in intact neuronal networks; electrophysiological recordings on individual neurons	Surviving neurons are completely functional; subsequent transfection of the same neuron with more than one construct at different time points; neurons up to 1 mm deep into a tissue can be transfected	Relatively time consuming; expensive equipment; relatively difficult to optimize	Moderate cell survival rate	Typically within hours; near physiological expression levels possible because physiological environment is maintained (depending on promoter used), therefore possible to image cells over months	No limit	No
Ca²⁺-phosphate co-precipitation	Cell types: Neuronal cell lines; differentiating and mature (fully differentiated) primary neurons in vitro Applications: Analyses requiring low numbers of transfected cells, e.g., live imaging of individual neurons in vitro; analyses depending on healthy neurons such as the assessment of developmental/morphological phenotypes in neurons, e.g. after siRNAi; covisualization of RNAs and proteins	Very cost-effective; no specialized equipment required; comparatively simple to optimize for a variety of plasmids; gentle method with minimal stress for the transfected cells (after optimization); amount of transfected DNA can be titrated to vary expression levels	Low transfection efficiencies for post-mitotic neurons (typically ∼5–10%), but can go up to 30% after optimization; transfection procedure can be relatively time consuming (progress of crystal formation and deposition on cells as well as cell viability may have to be monitored regularly over several hours)	Low (when crystal size and exposure time are optimized)	Typically within hours; depending on promoter and cell type: physiological expression levels within the first 12–18 h	No limit	No
Lipofection	Cell types: Neuronal cell lines; differentiating and mature (fully differentiated) primary neurons in vitro (and in vivo following injection of transfection solution) Applications: Transfection of a wide range of constructs and oligonucleotides; high transfection efficiencies (with little optimization) for cell lines; high efficiencies for RNAi knock-downs, also in mature neurons	Very simple and fast procedure with few optimization steps; suitable for transient and stable transfections; high reproducibility; cost-effective	Relatively low efficiencies for post-mitotic neurons (typically ∼1–5%), but can go up to 30% after optimization	Adverse effects on neuronal morphology and/or viability have been reported (depending on cell type and reagent)	Typically within hours; moderate to nearly physiological expression depending on promoter and cell type	No limit	No
Adenoviruses	Cell types: Cell lines and primary neuronal cells, including mature, fully differentiated neurons, in vitro; whole nervous system, including adult nervous system, in vivo Applications: Efficient in vitro and in vivo gene delivery, including expression of GOIs only in certain brain regions after localized inoculation with viral vectors; transient and inducible expression possible; suited for quantitative and biochemical analyses because of very high transduction efficiencies	Very high transduction efficiency in dividing and nondividing mammalian cells; no risk of insertional mutagenesis, as there is no genome integration	Labor intensive and expensive; safety issues (biosafety level 2 laboratory needed); immune/inflammatory responses in vivo; transduction of glia cells (can be limited with neuron-specific promoters)	High when high virus titers are used	Onset after a few days; high levels of expression that can last for weeks to even months	∼7.5 kb (high-capacity, helper-dependent AdVs: up to 34 kb)	No
Adeno-associated viruses	Cell types: Cell lines and primary neuronal cells, including mature, fully differentiated neurons, in vitro; whole nervous system, including adult nervous system, in vivo Applications: Efficient in vitro(cell lines, primary postmitotic neurons) and in vivo gene delivery; transient and stable transduction; transduction of neurons in brain slices; suited for quantitative and biochemical analyses because of very high transduction efficiencies; natural tropisms allow specific transduction of different cell types	Very high transduction efficiencies in dividing and nondividing mammalian cells; naturally replication incompetent/non-pathogenic; can integrate into the host genome	Labor-intensive and expensive; safety issues (biosafety level 2 laboratory needed); can cause immune/inflammatory responses in vivo; no site-specific integration into the genome with recombinant vectors; risk of insertional mutations	Low	Onset ∼2 weeks after transduction; high levels of expression	∼5 kb	Yes
Lentiviral vectors	Cell types: Cell lines and primary neuronal cells, including mature, fully differentiated neurons, in vitro; whole nervous system, including adult nervous system, in vivo Applications: Efficient in vitro(cell lines, primary postmitotic neurons) and in vivo gene delivery; transient and stable transduction and inducible expression; transduction of neurons in brain slices; suited for quantitative and biochemical analyses because of very high transduction efficiencies	Very high transduction efficiencies in dividing and nondividing mammalian cells easy to produce high-titer stocks and simple transduction procedure; no/little purification of viruses needed; low cell toxicity; integration into the genome; transduction of specific cell types, possible via pseudotyping of viral vectors	Labor-intensive and expensive; safety issues (biosafety level 2 laboratory needed); no site specific integration into the genome; possibility of insertional mutagenesis	Low	Few hours after transduction; high levels of expression	∼10 kb	Yes
Herpes simplex viruses	Cell types: Cell lines and primary neuronal cells, including mature, fully differentiated neurons, in vitro; whole nervous system, including adult nervous system, in vivo Applications: Efficient in vitro(cell lines, postmitotic primary neurons) and in vivogene delivery; tracing of neuronal pathways in vivo; transduction of neurons in brain slices	Natural neurotropism; very high transduction efficiencies in dividing and nondividing mammalian cells; no risk of insertional mutagenesis, as there is no genome integration; large insert size allows transduction of multiple genes or genomic regions	Labor-intensive and expensive; safety issues (biosafety level 2 laboratory needed); immune/inflammatory responses in vivo	High; lower with amplicon vectors	Few hours after transduction; high levels of expression, decreases within the first few weeks	>100 kb possible (with amplicon vectors)	No
Microinjection	Cell types: Large and robust neurons (neuronal cell lines; differentiating and mature primary neurons) in vitro Applications: Analyses requiring low numbers of transfected cells where specific cells are targeted, e.g. live imaging of individual neurons in vitro; introduction of molecules other than nucleic acids; injection into a specific subcellular region/compartment	Possible to inject substances that cannot be synthesized by a cell, e.g. labeled RNAs, neutralizing antibodies; transfection of specific cells or cell types in a mixed cell culture; possibility to inject into the nucleus (e.g. normal nuclear processing of RNAs)	Low transfection rates, limited by the features of the cell type (larger and more robust neurons are easier to inject and have a higher chance of surviving); very time consuming; relatively expensive equipment	Poor survival rate because of physical damage of neurons during injection	Expression plasmids: typically within hours; fluorescent signal of injected labeled RNAs very low	No limit	No
Biolistics	Cell types: All cell types in entire brains (in vivo) and tissue slices; cultured cells in vitro, including neuronal cell lines as well as differentiating and mature primary neurons (not suited for early differentiation stages however, as cells must be firmly adherent to substrate so as to not detach after bombardment with gold particles) Applications: Experiments on individual neurons (including mature neurons) in entire brains and spinal cords; relatively high transfection rates in vivo without the need for special safety measures; combined with two-photon microscopy: imaging of cells deeper inside the tissue; electrophysiological recordings on individual cells	Analyses on individual neurons in normal cellular context; quick protocol; neurons deep into a tissue can be transfected	Relatively expensive equipment and reagents; Relatively low transfection efficiencies (typically ∼2%); however, recently improved protocols lead to higher transfection efficiencies of up to 10% (cultured neurons) and up to 34% (slice cultures); collateral tissue damage in vivo	Significant cell damage caused by high pressure and accelerated gold particles; recently developed hand-held gene gun or use of ″mash″ show significant improvement	Typically within 1–2 d after bombardment; near-physiological expression generally persists for a minimum of 3–4 d in cell culture, up to 7 d in slices	No limit	No

↵^aIn addition to the parameters specific to each method, as described in this table, the onset, level, and duration of expression varies, e.g., with the plasmid and promoter used and the expressed construct, as well as the DNA concentration used.