Pii: s0959-4388(02)00365-3

Techniques for gene transfer into neurons
Philip Washbourne* and A Kimberley McAllister†
To illuminate the function of the thousands of genes that make varying sizes, including cotransfection with multiple up the complexity of the nervous system, it is critical to be able constructs; third, have limited cellular toxicity; and fourth, to introduce and express DNA in neurons. Over the past two be easy and safe to perform. Despite major advances in decades, many gene transfer methods have been developed, this field in the past several years, the ideal gene delivery including viral vectors, liposomes and electroporation.
system for all applications has yet to be developed. Although the perfect gene transfer technique for every Thus, the specific advantages and disadvantages of each application has not yet been developed, recent technical echnique must be considered in selecting a transfection advances have facilitated the ease of neuronal gene transfer method for any particular experiment [2] (Table 1).
and have increased the accessibility of these techniques to alllaboratories. In order to select a transfection method for any Because one ultimate goal of gene transfer lies in thera- particular experiment, the specific advantages and peutic remedies, much of the research into DNA delivery disadvantages of each technique must be considered.
to the nervous system is geared towards gene therapy.
However, reviewing the large and rapidly growing field Addresses
of gene therapy is outside the scope of this review; for Center for Neuroscience, University of California, Davis, gene therapy issues, including information on the use of 1544 Newton Court, Davis, California 95616, USA lentivirus in gene transfer, the reader is referred to several recent reviews and reports [3–9]. The objective of this review is, instead, to present an overview of neuronal Current Opinion in Neurobiology 2002, 12:566–573
transfection methods, to provide a few illustrative examplesof applications of these techniques, and to compare the 0959-4388/02/$ — see front matter 2002 Elsevier Science Ltd. All rights reserved.
most common methods for their suitability for gene transferinto postmitotic neurons in the central nervous system DOI 10.1016/S0959-4388(02)00365-3
Abbreviations
AAV
Recombinant virus-based technologies
Gene transfer into postmitotic neurons is a young field.
One of the first major breakthroughs in transfecting post- mitotic neurons came in 1988 with the demonstration of the first high-efficiency, virally mediated transfer of a foreign gene into neurons [10]. The increasing use of viral woodchuck hepatitis virus posttranscriptional regulatory element vectors for the transfer of DNA to neurons is undoubtedlydue to extremely high infection efficiencies (up to 95% of Introduction
neurons) compared with non-viral methods. This superiority A major challenge in current neuroscience research is to of virus-based systems comes as little surprise, because understand the functions of the thousands of brain-specific one is benefiting from what viruses have evolved to do — genes involved in neural development, plasticity, physiology, insert their DNA or RNA into host cells and express it.
and function. To accomplish this goal, we must have access This basic predisposition for infection makes viruses to techniques in which gene expression can be monitored relatively easy to use in both young and adult tissue and and manipulated in healthy cells, slices, embryos, and on such diverse preparations as dissociated cells, slices adult animals. Historically, transfection of postmitotic neurons has been labor-intensive, inefficient, unreliable,and/or cytotoxic. This inability to express foreign proteins Because many recombinant viral vectors are replication- in postmitotic neurons has, until the past few years, incompetent, most are also relatively safe to use.
hampered neuroscience research. Fortunately, a large Recombinant viral vectors can be locally applied or focally number of diverse techniques for transferring genes into injected into a group of neurons, either in culture or in postmitotic neurons have recently been developed and tissue, to produce highly localized expression of a gene of interest. However, these advantages are counterbalancedby some serious limitations — potential toxicity to neurons, It is now possible to express foreign genes in either a the effort and time to construct recombinant viral vectors, single neuron or a large population of neurons in dissociated limitations on size of the DNA expression cassette, and cultures, cultured slices, or in vivo. For basic research potential safety hazards to laboratory personnel [1,2,11,12••,13].
purposes, the ideal transfection method should: first, be There are a number of viral vectors currently being used to capable of transfecting postmitotic neurons with high transfect postmitotic neurons. These viral vectors differ in efficiency; second, allow transfection of constructs of terms of infection efficiency, expression levels, lag phase, Gene transfer methods for postmitotic neurons*.
h
niques for gene transfer into neur
Application:
dissociated
Information included in this table is based on current published reports. It is possible that results may vary depending on laboratory experience and, especially, the health of the neuronalpreparation. *A comparison of the most commonly used methods for gene transfer for postmitotic neurons. Please find references for each point in text. †Ratio determined using generalcytomegalovirus promoters; this could be changed by using neuron-specific promoters.
New technologies
and toxicity for the host cell or animal [12••,14,15•] organisms. In Xenopus laevis, vaccinia vectors have been (Table 1). Thus, the choice of viral vector depends greatly used successfully to transduce tectal neurons in dissociated cultures (H Cline, personal communication) and in vivo(see [32,33•] for examples).
Herpes simplex virus
The first virus to be used for gene transfer was herpes
Sindbis and Semliki Forest viruses
simplex virus (HSV) [10]. Neurons are a natural host for Recently, the related RNA viruses, Sindbis and Semliki HSV and expression of HSV-transduced genes can last for Forest virus (SFV) have received a lot of attention months to years. However, because of its cellular toxicity, [12••,34]. These viruses are selective for neurons (depending its difficulty to construct, and its high potential risk to on the strain) and can mediate recombinant protein humans, HSV is not commonly used [1]. Recent advances expression rapidly, reliably, and to high levels [12••,34].
in amplicon-based HSV vectors [16], decreases in toxicity, Relative to other viral vectors, they are less labor-intensive and increasing ease of use may allow these viruses to live thanks to commercially available kits (Invitrogen). Sindbis up to their early promise in the near future.
and SFV have been used with great success in vivo and in dissociated neurons and cultured slices (see [35,36] for Adenovirus
examples). In particular, Sindbis has been used to successfully Adenovirus (AdV) has historically been the most commonly transduce large numbers of hippocampal neurons in slices used viral vector, with applications ranging from gene transfer in vivo, to in vitro slices and dissociated neurons[17–20]. The first reports of recombinant AdV as an effective The potential major drawback to these viruses is that they gene delivery system for postmitotic neurons in vivo were shut off host protein synthesis within approximately 8 h of published in 1993 [21–23]. Expression begins a few days infection, leading to neuronal toxicity and death at variable following infection and lasts for weeks to months [11,12••].
times post-infection [1]. By carefully monitoring synaptic Although this vector can transduce postmitotic neurons in transmission, membrane potential, and input resistance, culture well [17], the success of recombinant AdV in trans- Malinow and colleagues have found that Sindbis infection ducing postmitotic neurons in intact tissue can be variable leads to significant toxicity only after 48 h (and probably [12••,17,18,20]. Furthermore, first-generation AdV is path- 72 h) post-infection in hippocampal slices (R Malinow, ogenic at high titers, transduces glia better than neurons, personal communication). Toxicity in dissociated neuronal is relatively difficult to construct, and can cause severe cultures arises approximately 24–48 h after infection immune reactions in vivo [1,2]. The new, second-generation, (J Sullivan, personal communication) and between 48 and helper-dependent, ‘gutless’ adenoviral vectors developed 72 h in vivo (R Malinow, personal communication).
in the past few years may alleviate these disadvantages[15•] and recent adenoviral vectors designed with neuron- Non-viral transfection methods
specific, inducible promoters are especially exciting [15•,24••].
Non-viral transfection methods comprise an eclectic mix ofchemical, physical and electrical methods for gene transfer.
Adeno-associated virus
Non-viral methods are advantageous for gene transfer into One of the most promising viral vectors is adeno-associated postmitotic neurons because they are generally easier to virus (AAV) [25]. In 1994, Kaplitt et al. [26] discovered that use, less toxic, and not constrained to delivering plasmids AAV vectors can selectively transfect neurons. AAV is the below a relatively small size (see Table 1 for comparison least toxic of all viral vectors, leads to high levels of gene with viral techniques). However, transfection efficiencies expression and has the potential for site-specific integration, resulting from non-viral transfection methods are generally leading to long-lasting gene expression. The limitations of considerably lower (except for electroporation) than effi- AAV vectors are two-fold: the recombinant protein starts to ciencies obtained with recombinant viral vectors [1] (Table 1).
be expressed after a delay of about two weeks post-infectionand the maximal insert size is only about 5000 nucleotides Chemical transfection methods
[11,12••,13]. Recently, AAV vectors have been used to The first subgroup of non-viral technologies, the chemical transduce postmitotic neurons in vivo, in dissociated primary transfection methods, includes calcium phosphate cultures, and in cultured brain slices [12••,13,25,27,28].
coprecipitation, liposomes, non-liposomal lipids such asEffectene (Qiagen), and high molecular weight cationic Vaccinia virus
polymers. Calcium phosphate-mediated transfection is one Vaccinia virus was one of the first viral vectors to be used of the oldest methods for gene transfer and is, along with successfully in transducing hippocampal slice cultures at lipofection, one of the most commonly used gene transfer extremely high efficiencies [29–31]. Recombinant protein methods for basic neuroscience applications. The physical starts to be expressed from 6–16 h post-infection [1]. In basis for this method is unclear, although it is believed that mammalian tissue, vaccinia quickly becomes highly toxic, the DNA-calcium phosphate coprecipitate enters the neuron causing 50% of transduced neurons to die within 18 h through endocytosis [1]. Although calcium phosphate following infection (R Malinow, personal communication).
coprecipitation has not been used to transfect neurons in However, this toxicity is not seen in non-mammalian intact tissue, it has been used extensively and successfully Techniques for gene transfer into neurons Washbourne and McAllister 569
Cis and trans cotransfection of fluorescently
tagged proteins into young dissociated
primary cortical cultures using lipofection
[52•]. This figure demonstrates cotransfection
of the same neuron with two constructs, or
neighboring neurons with distinct constructs.
Neurons were dissociated and cultured as
described [52•] and then transfected using
Lipofectamine 2000 (Gibco) at four days in
vitro. (a) Neurons were cis cotransfected with
a postsynaptic scaffolding protein
(postsynaptic density protein 95kDa
[PSD95]) linked to EGFP (PSD95–EGFP;
in green) and an N-methyl-D-aspartate
(NMDA) receptor subunit coupled to DsRed
(NR1–DsRed; in red). Both fusion proteins
are expressed in dendrites but show distinct
subcellular distributions in young cortical
pyramidal neurons. (b) Neurons were trans
cotransfected with growth-associated protein
43 (GAP43) — an abundant protein in growth
cones — coupled to EGFP (GAP43–EGFP;
cell body and proximal apical dendrite, but transfected into a neighboring neuron out of the illustrated field, is expressed in the axons dendrites, where it is highly expressed in the to transfect dissociated neuronal cultures from the CNS Several additional methods related to lipofection can and peripheral nervous system of many diverse species also be used to transfect postmitotic neurons. Effectene, a [41–44]. Cotransfection is also possible with calcium non-liposomal lipid produced by Qiagen, has been used to phosphate coprecipitation, leading to almost 100% cotrans- transfect dissociated neuronal cultures specifically to fection, although ratios of expression vary. The major achieve low levels of protein expression [56•]. High mole- drawback to this method is that transfection efficiencies cular weight polycationic polymers have also been used are highly variable but consistently low, in the range of successfully to transfect neurons [57]. Finally, immuno- liposomes or antibody-directed liposomes can begenerated by encapsulating liposomes with antibody- Despite reduced transfection efficiency compared to viruses, bound poly-ethylene glycol. These antibodies target the gene transfer using liposomes (lipofection) has had a complexes to specific cells, even across the blood–brain significant impact in many areas of neuroscience by virtue of barrier [58••], thus allowing brain-specific expression after its user-friendliness and versatility. Liposomes are positively intravenous administration. The importance of this charged lipid spheres with a diameter between 100 and method for gene therapy is striking and it should not be 500 nm [45•]. The surface positive charges on liposomes ignored by the basic neuroscience community, because attract the negative charges of both DNA and neuronal sur- immunoliposomes may constitute an inexpensive and less faces. In general, liposomes are believed to be endocytosed labor-intensive alternative to producing transgenic and by cells, although the precise mechanisms of DNA entry into the cell and transport to the nucleus are unknown [46]. Thecharge ratio and size of the liposomal particles strongly influ- Physical transfection methods
ence the efficiency and cell specificity of endocytic uptake The physical methods for transfection include micro- [45•,46]. Most recently, Invitrogen has developed a new mix- injection and biolistics. Microinjection involves directly ture of lipids called Lipofectamine 2000, which significantly injecting plasmid DNA into the nucleus of a neuron [59], increases the efficiency of neuronal transfection (routinely or injecting cRNA into the cytoplasm [60]. Whereas this 10–25%; PRMA Gomes and AK McAllister, unpublished method is standard for transfecting oocytes, Xenopus data). Since the first description of lipofection in 1987 [47] blastomeres (see [53•] for example), and invertebrate and its first use in vivo in 1990 [48], lipofection has been used neurons, it requires considerable skill with mammalian in several different applications in vitro [49–51,52•] (Figure 1) CNS neurons and has not become a routine approach.
and in vivo [53•,54]. Recent attempts to improve on the Microinjection is quite labor-intensive and can be used on transfection efficiency of lipofection have led to the only a small number of neurons at a time. However, for discovery that anionic liposomes largely increase transfection applications in which only one identified neuron needs to efficiency of oligonucleotides in neurons [55•], but it remains be transfected, this method can be used effectively and to be seen whether this will be made commercially available.
New technologies
Biolistics, short for biological ballistics, involves bombarding Transfecting neurons in slices is optimal using either viral neurons at high velocity with DNA-coated gold particles vectors to transiently transduce large groups of neurons [62,63]. Neurons whose nuclei are penetrated by a gold [12••,19,24••,31,37,38,40] or biolistics to achieve a large particle have a high likelihood of becoming transfected.
number of healthy, dispersed transfected neurons with Transfection efficiencies are relatively low in dissociated long-lasting expression [63,71]. Viruses are particularly cultures (1–5%), but higher in cultured slices (up to several effective in transducing the large number of neurons hundred neurons per slice) [62]. Biolistics is straight- necessary for biochemical analysis [38]. Finally, transfecting forward and reliable but requires optimization to minimize neurons in vivo has recently become much more successful physical damage to cells or tissue and investment in a gene using exciting new modifications to electroporation gun (BioRad). Although biolistics has not, to date, been [65•,70••] and viruses [15•,32,33•,39•].
successful in transfecting neurons in vivo, it is particularlyuseful for transfecting neurons in a dispersed manner in Technologies for transfecting postmitotic neurons have slices and primary cultures [63,64].
vastly improved in the last five years, providing basicresearchers with many options and allowing experiments Electrical transfection methods
to be performed that were, until recently, technically Perhaps the most promising non-viral method for trans- impossible. The field of neuronal gene transfer for basic fecting postmitotic neurons is electroporation. Although research applications is currently focused on two major the physical basis for this method is unknown, it is issues — improving transfection efficiencies and targeting believed that electric shock transiently opens pores in the genes to specific neuronal types. The first goal — to cell membrane, allowing charged molecules to enter cells improve transfection efficiencies — is steadily being by electrophoresis [65•]. In the past, this method has been achieved through rapid advances in both viral and non- limited by the damage caused by these electrical pulses; viral transfection technologies. Recent reports suggest that however, recent advances have dramatically improved combining viral and non-viral approaches may allow neuronal health. Unlike the other non-viral transfection researchers the best of both worlds [72,73]. The second methods, electroporation results in large numbers of goal for the field is to develop ways in which near-endogenous healthy, highly expressing transfected neurons. Single cells expression levels and specific transfection of neuronal to entire tissues can be transfected with single or multiple subtypes can be achieved. Currently, most transfected genes constructs by varying the size of the electrodes and are driven by the ubiquitous and powerful cytomegalovirus modifying the pattern of stimulation. In fact, in vivo promoter. However, neuronal specificity of transfection electroporation is now routinely used by both chick and can be increased by using neuron-specific promoters [74], mouse embryologists [66–68]. Electroporation has also such as the platelet-derived growth factor β-chain promoter been adapted to transfect dissociated neurons in culture [12••] or the synapsin 1 promoter [24••,75••], and the timing [69]. Perhaps most exciting, Cline and colleagues have of expression can be controlled by using neuron-specific, developed a new method to target gene transfer to single inducible promoters [24••]. Thus, recent advances in trans- neurons in vivo using single-cell electroporation [33•,70••].
fection technologies are making it possible to address the Electroporation is also the most versatile of the non-viral functions of proteins in neuronal development and adult- technologies; it can be used not only for gene transfer, but also potentially to target any charged macromolecule toneurons including dyes, drugs, antibodies, antisense Acknowledgements
oligonucleotides, double-stranded RNAs, and bacterial or We thank Holly Cline, Robert Malinow, Sam Young, and Jane Sullivan for yeast artificial chromosomes [65•].
critical information on recombinant viral methods and Leon Hall, Karl Murray,and Marty Usrey for informative discussions and critical reading of themanuscript. Our research is supported by the Alfred P Sloan and Pew Conclusions and future directions
Foundations (AK McAllister), the March of Dimes (AK McAllister) andNational Institute of Health grant RO1 EY13584 (AK McAllister).
Recent advances in technologies for gene transfer to post- P Washbourne is a Medical Investigation of Neurodevelopmental Disorders mitotic neurons present neuroscientists with an abundance of methods, each with their individual advantages and disadvantages (Table 1). Thus, researchers must choose a References and recommended reading
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