Genetic targeting of specific neuronal cell types in the cerebral cortex



            Understanding the structure and function of cortical circuits requires the identification of and control over specific cell types in the cortex. To address these obstacles, recent optogenetic approaches have been developed. The capacity to activate, silence or monitor specific cell types by combining genetics, virology and optics will decipher the role of specific groups of neurons within circuits with a spatiotemporal resolution that overcomes standard approaches. In this review, the various strategies for selective genetic targeting of a defined neuronal population are discussed as well as the pro and cons of the use of transgenic animals and recombinant viral vectors for the expression of transgenes in a specific set of neurons.

 Keywords: optogenetics, opsins, transgenesis, recombinase, aaV, neuronal network.


1. Introduction

Of all brain structures, the cortex, which represents over 80% of the volume of the human brain, is what makes us human. Paradoxically, the basic local architecture of the neocortex is quite similar in all mammals, from mouse to man, comprising cortical columns composed of several neuron types organized in units (Hubel, 1977). It is estimated that the human cortex contains two millions of these cortical modules, each of which composed of 10,000 to 70,000 neurons. These local cortical modules are organized in a framework with a six-layered architecture, in which neurons with distinct functional properties are distributed in discrete layers.

In order to understand how the brain works, it is necessary to experimentally modulate the activity of neuronal circuits in a highly specific and temporally controlled manner. Electrical or pharmacological stimulation of neuronal networks were extensively used in the past; today, the recent developments of optogenetics greatly extend our ability to decipher neural circuits.

Optogenetic tools are divided into two families referred as optogenetic actuators and optogenetic sensors: actuators are suitable for controlling specific neuronal populations and are described in chapter X; sensors are suitable for monitoring neuronal networks. Optogenetic sensors are described in details in chapter X.

Briefly, optogenetic actuators are based on opsins that are seven-transmembrane proteins containing the light-isomerizable chromophore all-trans-retinal. When illuminated, these proteins can rapidly translocate ions across the membranes of the cells in which they are expressed.

Several features have made actuators of prime importance for studying the cortex. First, light activation has a higher spatio-temporal resolution than electrical or pharmacological brain stimulation. Second, it has been demonstrated that actuators could be used in living cells with minimal toxicity (Zhang, 2007). Third, they can be fused to fluorescent proteins without loss of activity, so it allows visualization of correct expression in cells. Last but not least, actuators are proteins, so their expression can be selectively restricted to certain cell types and/or at specific locations. As a result of these unique properties, actuators and sensors have been used in an increasing number of studies to control neuronal networks in a large number of species both in vitro and in vivo.

One of the biggest challenges for optogenetics is to genetically modify a chosen population of cells for expressing the light sensitive proteins. In this review, we will first explain how to identify molecular markers (enhancer/promoter) specific to a given cortical cell type. Then we will overview the various methods to deliver genes coding for opsins by the development of transgenic animal lines or direct transfection of neurons. We will go ahead by highlighting the available strategies to express more selectively and more efficiently the opsins in the targeted cells using regulation of the transcription or recombinase-based conditional systems. Finally, we will provide a summary of the technical progress in viral mediated gene delivery, with an emphasis on new strategies to label specific population with high specificity.

2.     Targeting cell types by controlling gene expression

As it is possible to characterise populations of neuron that express specific molecular markers, it is possible to use cis-regulatory elements controlling the expression of these markers to genetically target a desired protein. Here we present the general concepts to regulate gene expression and how transgenesis of optogenetic actuator/sensors could be used to control and monitor specific neuronal populations in the brain.

2.1.     Neuronal cell types: from phenotype to genotype

The cortex is constituted of several different cell types such as neurons, astrocytes, microglia, oligodendroglia and epithelial cells which are mixed in a highly complex three dimensional structure. Here we will focus on neurons. To understand how the brain processes information, we must understand the structure of its neural circuits, defined as functional entities of interconnected neurons that influence each other. On a local level, the function of a neuron derives from its morphological, electrophysiological molecular properties and its embryonic origin. Even if these parameters are implemented by rules that are only partially understood, it is reasonable to assume that a phenotypic network architecture gradually emerges from the execution of the various developmental instructions encoded in the genome. In this chapter we will present molecular parameters commonly used to classify neurons and demonstrate that a defined cell population can be characterized by a specific pattern of gene expression.

As our understanding of the vast diversity of neurons in the cortex develops, it has become clear that one or more molecular markers should be sufficient to define a specific neural populations. In fact, for both pyramidal cells and interneurons, global expression profiling can help grouping neurons into classes that reflect meaningful biological properties (Cauli, 1997, Karagiannis, 2009, Subkhankulova, 2010). Although the full transcriptional map at single cell resolution is not yet achievable, a lot of progress has been made in identifying a number of the relevant genes based on single cell RT-mPCR (Lambolez, 1992). If excitatory cells express only a limited set of specific molecular markers (Kubota, 1994), different types of inhibitory interneurons might be distinguished by a single marker or by a combination of markers (molecular profile) including transcription factors, neurotransmitters, neuropeptides, calcium binding protein, receptors (iono/metabo-tropic), structural proteins, cell surface markers, ion channels, connexins, transporters and more (Ascoli, 2008).

Today, there is no single morphological, electrophysiological, or developmental parameter that is able to specifically describe a neural subtype. The regulation of gene expression within a specific cell type is spatially and temporally defined. We thus assume that molecular markers whose expression are controlled by specific promoters are suitable to group classes of neurons. In order to restrict the expression of optogenetic trangenes to a class of neurons, the transgenes are incorporated under the control of specific promoters activated only in a class of neurons. In conclusion optogenetics is based on expression of genetically encoded actuators and are becoming a powerful tool for deciphering neural circuits as described in the following part of this review.

2.2.     Cis-regulatory elements (Promoter/Enhancer)

       The factors controlling gene expression are complex because the ability to produce biologically active proteins comes under regulation at DNA, RNA and protein levels. Nevertheless, the control of transcriptional initiation is one of the most important regulation modes.  For a gene to produce a protein it requires a promoter. A promoter is a section of DNA in front of the gene that functions to recruit the cellular machinery that will initiate the multi-step process of protein production. Enhancer is a short region of DNA that can modulate transcription of genes. They consist of several composite elements or/and individual binding sites for transcription factors (TFs) but their position in respect to transcription start are different. A promoter can roughly be divided in two parts: a proximal part, referred to as the core, and a distal part. The proximal part is believed to be the regulatory part of the gene that promotes recognition of transcriptional start sites by RNA polymerase and that is responsible for the basal level of transcription (Berk, 1999, Nikolov, 1997). It is mediated by elements, such as the TATA and Initiator boxes through the binding of the TATA box-binding protein, and other general transcription factors specific for RNA polymerase II (Featherstone, 2002). Transcription factors are molecules involved in regulating gene expression. They are usually proteins, although they can also consist of short, non-coding RNA. Transcription factors are also usually found working in groups or complexes, forming multiple interactions that allow for varying degrees of control over rates of transcription. In eukaryotes, genes are usually in a default "off" state, so transcription factors serve mainly to turn gene expression on. The distal part of the promoter includes elements that regulate the spatio-temporal expression (Fessele, 2002, Tjian, 1994). In addition to the proximal and distal parts, regulatory regions that contain enhancer and/or repressors elements have also been described (Bagga, 2000, Barton, 1997). They modulate the level of transcription depending on the type of tissue, developmental stage, stage of the cell cycle, induction by hormones or other molecular signals. Positions of enhancers can vary significantly within the gene as they could be located just before the promoter or at a distance of several thousand base pair in the 5' region, within introns, or in 3'-regions (Kadonaga, 2004). Lastly, eukaryotic genomes can be organized into domains of transcriptional activity or transcriptional silencing, encompassing one or more genes (Oki, 2002).

             Even if the regulatory motifs of some specific genes have been investigated in detail, there are not yet clear and unequivocal descriptions of genomic segments that contain all elements required to activate transcription. Nevertheless, to increase scientific community access to general information about promoters and their functions, regulations, there are many specialized databases such as the Eukaryotic Promoter Database (EPD) containing description about promoters, as they are defined by an experimentally proven transcription start site and their tissue-specificity (Perier, 1999).

3.     Genetic modification of host genome by transgenesis approaches

3.1.     Germline transgenesis

As discussed in the previous chapter, the cis-regulatory elements can be hundreds of kilobases pairs long and are thus difficult to manipulate both in vitro and in vivo. However, many strategies have been extensively developed in mouse because of the powerful genetic tools that are available to manipulate the mouse genome and to integrate large DNA fragments into the host genome. Actually two different strategies could be applied to generate a transgenic mouse termed random or positional transgenesis in relationship with the control site of transgene integration. A transgenic construct requires three main components: 1) a promoter to drive expression of the transgene, 2) a transgenic open reading frame encoding gene to be expressed and 3) a polyadenylation signal to terminate transcription. Bacterial artificial chromosome (BAC)-mediated transgenic fulfil these criteria as BAC vectors can accommodate large genomic fragment up to 700 kb that may contain several contiguous genes with their cis-regulatory elements (Shizuya, 1992). BACs offer several advantages because they are easy to manipulate in vitro and their integration is generally stable (Marra, 1997) with a linear relationship between the copy number and the level of expression of the integrated gene (Chandler, 2007). In addition, BACs can be modified by homologous recombination in E. coli, to introduce desirable mutations including insertions, deletions and point mutations (Gong, 2002).

3.1.1.     Random transgenesis

Random transgenesis (Figure 1A) is the most rapid and effective method to generate transgenic mice. The main technical step is the direct microinjection of BAC DNA into the pronucleus of fertilized mouse eggs, followed by transfer of the injected oocytes to pseudogestant foster mothers. Pups that arise from a transfer have the foreign DNA stably and randomly integrated into the genome and can then germline transmit the integrated transgene to their offspring to establish a transgenic mouse line. BAC DNA constructs are large enough to contain all the regulatory elements necessary to confer accurate transgene expression in vivo. The major advantage of using BAC transgenesis is to overcome positional effects (Heintz, 2001), meaning that the integration site has little or no effect on the expression of the transgene. Random transgenesis is an effective and efficient method to produce genetically engineered mouse models as demonstrated in GENSAT project that makes available to researchers with a collection of around 600 mouse lines expressing GFP under a particular promoter (Heintz, 2004). Since the transgene integration is a random event, each transgenic line is unique because the transgene is integrated at a distinct chromosomal location and with a definite number of copies. Rather than recapitulating the activity of each promoter/enhancer, expression of the target gene is often limited to a subset of cells in which cis-regulatory elements are active. Thus, different transgenic lines made with the same transgene selectively label different highly restricted subsets of neurons. For example, random insertion of the CamKIIa promoter, which is normally expressed in most excitatory forebrain neurons can be restricted to specific cell types of the striatum and hippocampus (Kellendonk, 2006, Nakazawa, 2002, Tsien, 1996). In fact, in many cases transgenes are integrated as multiple copies from one to several hundred that usually form head-to-tail repeats. Because of these limitations (unknown location in the genome and copy number) it is often necessary to perform phenotypic studies on several transgenic lines that have been generated. Even if BAC transgenic animals contain a high number of transgene’s copies, the amount of protein in each cell is dependent on the strength of the promoter. Because most of the promoters are relatively weak, this strategy could be non suitable for optogenetic tools as they require a high expression level. Nevertheless, a number of mouse strains expressing actuators have been generated (VChR1, eNphr3.0 for more details see One recent success is the generation of healthy ChR2-eYFP transgenic mouse with expression throughout the brain using the Thy1 promoter (Arenkiel, 2007, Zhao, 2008). Thy1 is an immunoglobulin superfamily member that is expressed in projection neurons in many parts of the nervous system, as well as by several non-neuronal cell types, including thymocytes (Gordon, 1987). Under these conditions, ChR2 can be used to map synaptically connected neurons in slices and even in anesthetized mice (Arenkiel, 2007, Wang, 2007). Several transgenic mice expressing opsins with specific neuronal targeting have now been generated (Hagglund, 2010, Thyagarajan, 2010).

3.1.2.     Enhancer and Repressor Trap

As the characterization of cis-regulatory elements of each gene is difficult to perform, alternative strategies based on random integration have been developed. An example is the enhancer trap (Figure 1B) approach that uses positional effects dependent on transgene insertion-site in the host genome. Short promoter segments from marker genes can restrict expression to the population of cells expressing the marker and positional effects can further restrict expression within that population. The power of this strategy was revealed following detailed analysis of lines in which highly restricted subsets of neurons were labeled by fluorescent proteins under the control of a short segment of the Thy1 promoter (Feng, 2000) or the Gad promoters (Chattopadhyaya, 2004, Lopez-Bendito, 2004, Oliva, 2000). This method has been also successful in flies (Bellen, 1989) and zebrafish (Davison, 2007, Nagayoshi, 2008, Scott, 2009).

On the contrary to the enhancer trap, another strategy called repressor trap could be used to inhibit expression of the transgene (for review see (Luo, 2008). In this case, the structure of the chromatin close to the integration site can affect the ability of transcriptional regulatory proteins and RNA polymerases to find access to specific genes and to activate transcription. Two primary mechanisms exist that alter chromatin structure and as a consequence affect alterations in gene expression. These mechanisms are methylation of cytosine residues in the DNA that are found in the dinucleotide, CG referred as a CpG dinucleotide and histone modification.

3.1.3.     Positional transgenesis

Positional transgenesis (Figure 1C) often termed knock-in gene targeting is the most faithful system to mimic endogenous gene expression. As randomly integrated transgenes are susceptible to silencing genes, targeting transgenes to a chosen location in the mouse genome has a number of advantages (Bronson, 1996, Misra, 2002). Firstly the integration site can be chosen to allow insertion of the transgene into a region of chromatin favourable for expression and that avoids an undesirable insertional mutagenesis. Additionally only a single copy is introduced which avoids problems associated with large multicopy arrays. This strategy requires embryonic stem (ES) cells that are harvested from the inner cell mass of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes. The introduction of the gene into ES cells is a multiple step process. Targeting constructs are introduced into the ES cells by electroporation. Then, the cell undergoes homologous recombination at the shared sequence, during which two crossover events replace the WT gene with the targeting construct. Correctly targeted cells inherit an antibiotic resistance gene and are able to grow in the presence of that antibiotic. The presence of the desired mutation in the embryonic stem cell DNA is then directly confirmed by PCR or genomic southern blot. ES cells with one copy of the desired mutation (heterozygous) are expanded, injected into blastocysts harvested from mice with black coat color, and implanted into pseudogestant females. Chimeric offspring in part deriving from the ES cells (dominant agouti coat color) and in part from the donor blastocysts (black coat color), are identified by patchy agouti/black coat color. Subsequent crosses will be made until the desired genotype is obtained. ES cells can thus be used as a vehicle to obtain transgenic mice from germline ES cell-mouse chimeras. One additional advantage of positional transgenesis is the ability to pre-screen ES cell clones for expression (Bronson, 1996). This strategy has also its limitations including the limited size of genomic DNA that can be inserted. In addition, DNA fragments smaller than 20kb often result in positional effects including lack of transgene expression, expression restricted to only a subset of cells or extinction of transgene expression in successive generations. Moreover, availability of ES cells is limited for a number of mouse strains (i.e. 129SV, C57/bL6J). Positional transgenesis is usually more time consuming and costly compared to random mutagenesis because of technical manipulations required for insertion of drug selection cassettes (Testa, 2003), ES cell culture, generation of ES cell-mouse chimeras and germline breeding. This strategy has been successfully applied to express ChR2 in GABAergic neurons (Katzel, 2011).

3.1.4.     Integration in a specific locus

At best, targeting the site of integration by homologous recombination in embryonic stem cells would virtually solve any chromosomal position effects since the transgenic construct would then be controlled by all regulatory sequences present in the chosen endogenous locus. Alternatively, particular locations in the host genome can be selected according to their capacity to allow adequate expression patterns of experimental transgenes (Wallace, 2000).

ROSA26 locus

This strategy has been successfully applied in mice since the discovery of the Gt(ROSA)26Sor locus (ROSA26). The ROSA26 locus (Figure 1D) was first isolated in a gene-trap mutagenesis screening performed in mouse embryonic stem cells (Friedrich, 1991). The ubiquitous expression of ROSA26 in embryonic and adult tissues, together with the high frequency of gene-targeting events observed at this locus in murine ES cells has led to the establishment of several ROSA26 knock-in lines. These represent a variety of transgenes including reporters (Soriano, 1999), site-specific recombinases and recently optogenetic actuators (hChR2(H134R)::tdTomato and hChR2(H134R)::YFP from H. Zeng, Allen Institute for Brain Science).

Hipp11 locus

As transgenic strategies are in constant evolution, the new Hipp11 (H11) locus (Figure 1D) will probably replace ROSA26 in the future because homozygous insertions into this locus are not predicted to disrupt any endogenous genes and the resulting mice are completely healthy and fertile (Hippenmeyer, 2010).

HPRT locus

Recent studies have also shown that it is possible to take advantage of both random and positional transgenesis method to insert BAC as a single copy at a specific genomic location such as hypoxanthine phosphoribosyltransferase (HPRT) locus and thus express transgene with the appropriate tissue and cell-specific pattern (Heaney, 2004, Miyazaki, 2005). However as the HPRT locus is on the X chromosome it will be randomly inactivated in female mice, which may not be ideal for every experiment.

attB site

Recently, it has been demonstrate that an intact single-copy transgene can be inserted into predetermined chromosomal locus containing attB site with high efficiency and faithfully transmitted through generations by FC31 integrase approach (Figure 1E). This system allows production of transgenic mice with the advantages of random transgenesis via pronuclear injection and precision of positional transgenesis (Tasic, 2011).

3.2.     Somatic gene transfer into the brain by DNA electroporation

       As mentioned before, the specific factors that exert control of gene expression include the strength of promoter elements, the presence of enhancer sequences and the interaction between multiple activator and inhibitor proteins. All these elements are encoded in DNA and could therefore be used to target optogenetic tools into defined group of neuron. Electroporation has become a common laboratory technique for enhancing the efficiency of DNA delivery into cells. Application of low voltage rectangular pulses after local injection of DNA temporarily increases in the trans-membrane potential difference, which provokes cell membrane permeabilization and facilitates DNA uptake. Developed initially on chick embryo (Itasaki, 1999), in vivo electroporation has now been successfully applied in several model systems for the delivery delivery of genetic materials (Isaka, 2007).

If performed in embryonic mouse, in-utero in vivo electroporation provides a powerful tool for the manipulation of neurons in the cortex and allows for the targeting of specific neuronal layers. Technically, DNA is injected in the lateral ventricles of the developing embryo and electroporated into neuronal progenitors lining the walls of the lateral ventricle. DNA electroporation in mouse was initially restricted to pyramidal cells (Saito, 2001). Additional technical developments  made it possible to specifically target gene expression to interneurons by in utero electroporation directed to the ganglionic eminences (Borrell, 2005). With stereotaxic apparatus facilitated microinjection, in vivo electroporation could also target a defined small area in the adult brain (Wei, 2003). Optogenetic tools have been successfully electroporated into mouse embryos (Adesnik, 2010, Gradinaru, 2007, Lewis, 2009, Petreanu, 2007).

Recently, electroporation has been refined to allow delivery on a single neuron scale both in vitro (Teruel, 1999, Mertz, 2002), in slices (Haas, 2002) and in vivo (Judkewitz, 2009). Single neuron electroporation presents many advantages such as the transfection of multiple genes using plasmids at the same time or the labelling of very few neurons at precise locations within the brain. As an example of such strategy for optogenetics, the expression of ChR2 has been successfully used to activate sparse pyramidal neurons in barrel cortex (Huber, 2008).

            Even if electroporation requires very specific technical skills, this strategy provides high expression levels that are needed when using bacterial opsins and thus is optimal for development of new optogenetic constructs as it requires less time (2-5 weeks instead of several months) than transgenic approaches. Moreover, the size of the DNA used for in utero electroporation is not limited which allows for a very specific targeting of cortical neurons. Another advantage is that opsins are expressed early, which makes possible electrophysiological studies in brain slices at young age. Nevertheless, there are a number of limitations including the restricted spatial expression of optogenetic probes (especially in mouse where labelled cells are mainly pyramidal cells of the cortex) and the variability of expression level among experiments.


4.     Genetic strategies for refined gene targeting

Over the past decades, an expanding repertoire of genetic tools has greatly facilitated the visualization and manipulation of cell populations in model organisms. We will describe here the general concept of driving the expression of a target transgene by using two (binary expression) or more (combinatorial expression) promoters that could be used following the Boolean logic gates AND (expression is only possible by the intersection of two expression patterns i.e. in a population of cells expressing A and B) or NOT (expression is exclusively restricted in one pattern).

4.1.     Binary expression strategies

The most versatile strategies for cell type specific targeting are binary systems, which uses the natural cis regulatory elements of endogenous genes to drive the expression of a primary effector, usually a transcription factor (Figure 2A) or recombinase (Figure 2B). This primary effector then activates expression of the transgene encoding a secondary effector that allows the expression of the transgene of interest.

4.1.1.     Control of transcriptional activity

Ectopic expression has proved to be an excellent technique for analysing gene function in Drosophila and other model organisms as demonstrated by the GAL4 system allowing the expression of any given open reading frame. The GAL4 system was built on the characterization of transcriptional regulation in yeast. GAL4 is an archetypal eukaryotic transcription factor isolated as an activator of the genes responsible for galactose metabolism in Saccharomyces cerevisiae (Hashimoto, 1983). The target sequence of GAL4 was defined as a 17-mer, four copies of which are found in the upstream activation sequence (UAS) of the galactose metabolism genes, Gal10 and Gal1 (Bram, 1986, Giniger, 1985, Webster, 1988). Furthermore, the activity of GAL4 is repressed by a physical interaction with the GAL80 protein, which is repressed when galactose is the only carbon source (Lue, 1987, Wu, 1996). The high level of conservation in the eukaryotic transcriptional machinery means that GAL4 can activate transcription in other species, as distantly related as humans and plants (Kakidani, 1988, Ma, 1988, Webster, 1988). The generation of GAL4 transgenes under the control of short promoter/enhancer fragments offer a powerful strategy for labelling more restricted subsets of cells. By using an artificial promoter that contains a tandem array of GAL4 binding sites and a transcriptional start site, expression of a target gene can be controlled by the expression of GAL4 (Figure 2A). The advantage of this system is that simply expressing GAL4 in a different tissue can change the expression pattern of the target gene. This system has multiple applications, especially when a large collection of different tissue specific GAL4 expressing transgenes is available. Thus, besides the use as a flexible system to express genes in different tissues, it can also be used to generate conditional overexpression lines, for targeted ablation of cells by expression of toxins and for tissue specific RNAi. GAL4/UAS has been successfully used in combination with the enhancer trap screens in flies what led to characterization of almost 7000 GAL4 lines (Hayashi, 2002). This system has also been used in mouse (Ornitz, 1991) and in zebrafish (Scott, 2007).

            Another binary expression system available in fruit fly is based on the repressor LexA that is a regulator of the SOS response to DNA damage in Escherichia coli (Walker, 1984). LexA is a two-domain protein including a DNA-binding domain and a dimerization domain. LexA binds as a dimer with varying affinities to single or multiple copies of gene-specific LexA DNA-binding motifs (Lex Aop) found upstream of its target genes. Fusing the C-terminal activation domain derived from various eukaryotic transcription factors to LexA allows it to drive in vivo the transcription of reporter transgenes in Drosophila whose promoters contain LexAop motifs (Lai, 2006, Szuts, 2000).

Recently, a new repressible binary expression system based on the regulatory genes from the Neurospora quinic acid gene cluster has been reported (Potter, 2010). This Q system offers many applications for labelling more restricted subsets including combinatorial logic gates with the GAL4 system as described in chapter 3.2.

4.1.2.     Site-specific recombination approach


The site-specific recombination system is also a binary system. The system begins with the cre gene, short for cyclization recombination, which encodes a 38 kDa recombinase protein from bacteriophage P1 that mediates intramolecular (excisive or inversional) and intermolecular (integrative) site specific recombination between loxP sites (for review see (Sauer, 1993)). These sites are known as loxP (locus of X-over P1) sequences, which are 34 base pairs long (two 13 bp inverted repeats separated by an 8 bp asymmetric spacer region) and are recognition sites for the Cre to recombine the DNA surrounding them. One molecule of Cre binds per inverted repeat or two Cre molecules line up at one loxP site. The recombination occurs in the asymmetric spacer region. Those 8 bases are also responsible for the directionality of the site. Two loxP sequences in opposite orientation to each other invert the intervening piece of DNA; two sites in direct orientation dictate excision of the intervening DNA between the sites leaving one loxP site behind. The Cre/lox system is a bipartite system in which one transgenic line, the driver, expresses Cre recombinase in a known temporal and spatial pattern and a second transgenic line, the reporter, contains a Cre recombinase dependent transgene that is under the control of an ubiquitous promoter. This reporter line contains a transcription stop flanked with two loxP sites in the same orientation even termed a “floxed stop” or LSL (lox-stop-lox) sequence. The stop sequence is a short sequence with several transcriptional stop codons that will prevent the gene from producing a protein. When Cre is present in the cells of this organism, it catalyzes recombination between the loxP sites, thereby deleting the stop sequence and allowing the expression of the target gene (Figure 2B). Therefore, if the Cre gene is bound to a promoter that only allows Cre production only in neuronal cells, the target gene will be specifically expressed in those cells.

Since the Cre/lox system has been extensively used over the last fifteen years, there are now numerous transgenic animal, plant and bacterial stocks that already contain the cre gene driven by ubiquitous or tissue-specific promoters. For example, there is an extensive collection of Cre driver mice from the NIH Neuroscience Blueprint that were initially designed for the tissue and cell-type-specific perturbation of gene function in the nervous system. (e.g.,; (Gong, 2007, Gong, 2002, Heintz, 2004). Cre driver lines in combination with “floxed stop” transgenic lines are an invaluable system that has the advantage of working in almost any type of cell. This strategy will probably be used in the future to express ChR2 in a specific cell types, using the JAX Rosa-CAG-LSL-ChR2(H134R)-EYFP-WPRE transgenic strain from H. Zeng.

Nevertheless, there are some disadvantages of the Cre/lox system. First, as mentioned in the chapter 2.1 it could be difficult to identify a promoter that is perfectly specific for a class of cells (Sauer, 1993). Second, the Cre recombination is an irreversible process that can produce “non-physiological” response when the driver promoter is transiently activated. This limitation has been overcome by to the development of inducible Cre (see chapter 4.3.). Last but not least, the establishment of transgenic systems with inserted genes requires a significant amount of time and money.


Another similar recombination strategy came from the yeast in the form of Flippase/FRT analogous to the Cre/Lox recombination system. The 2 micron plasmid (2µm) of Saccharomyces cerevisiae codes for a site-specific recombinase, the Flippase recombination enzyme (Flp), that catalyzes efficient recombination across two 599-base-pair inverted repeats (referred as Flippase Recognition Target (FRT) sites) of the plasmid DNA both in vivo (Broach, 1982) and in vitro (Vetter, 1983). Flp/FRT has been used to control gene expression by “FLP-out”: a recombinase-catalyzed intramolecular excision of spacer DNA including a transcriptional stop between tandemly oriented FRT sites. As for Cre/lox system, the gene downstream of the spacer is not transcribed until activation of the FLP recombinase and subsequent FLP-out (Golic, 1989, Struhl, 1993). After the FRT-containing cassette is excised by the FLP recombinase, the downstream gene is brought into proximity to the ubiquitous and constitutively active actin promoter and is therefore expressed (Figure 2B). This system and related systems have proven quite powerful and flexible in model organisms including Drosophila (Struhl, 1993) and mouse (Dymecki, 1996).


More recently, a screen of recombination systems derived from the resolvase/invertase family for site-specific recombinase activity in the fission yeast Schizosaccharomyces pombe has highlighted seven new recombination systems (Thomson, 2006) that could be used independently or in combination with other binary expression systems to increase specificity of transgene expression.

4.2.     Combinatorial expression strategies

Despite the success of binary expression strategies, the small number of genes with a highly restricted expression might limit its efficiency. Even the most restricted marker genes are likely to be expressed by multiple cell types. As an example, it has been demonstrated that the calcium binding protein parvalbumin that is a specific marker for fast spiking interneurons is also expressed in some of layer V pyramidal cells in barrel cortex (McMullen, 1994, Tanahira, 2009). To restrict with a higher precision expression of a transgene, is it possible to use combinatorial approaches. In these strategies, expression of a genetic marker is dependent on the presence of two factors, each of which is restricted to a subpopulation of neurons. Therefore, expression is more specific because it only occurs in cells that are in the intersection of the two populations.

4.2.1.     Intersectional strategy

Cell types are typically defined by expression of a unique combination of genes, rather than a single gene. Intersectional methods are important to selectively access specific cell types with a higher resolution. To achieve this goal, one possibility is to modify previously described binary strategies (i.e. Cre/loxP system) by replacing the ubiquitous promoter with a second tissue-specific promoter, such that the transgene of interest can only be expressed in cells in which both promoters are active.

4.2.2.     Dual binary system

A useful way to access specific cell types with precision depends on combinatorial restriction of transgene expression by independently targeting the recombinases Cre and Flp using distinct promoters (Awatramani, 2003). In this dual recombinase system, expression of the transgene of interest is made contingent upon the excision not of one but of two stop cassettes placed between the transgene and a broadly active promoter. Each stop cassette is flanked by target sites for only one of the two recombinases so that only in cells expressing both Cre and Flp will both cassettes be removed, allowing transcription of the transgene of interest (Figure 2C)

One approach to refining spatial regulation of the GAL4 system is to combine it with the FLP-out technique (Struhl, 1993). In this strategy, a terminator cassette flanked by FRT sites is inserted between the UAS promoter and the gene to be expressed, rendering the transgene silent. Activating the transgene requires the expression of the Flp recombinase to remove the terminator cassette (Figure 2D). The use of a heat shock inducible hsFLP recombinase affords temporal control of the onset of transgene expression. A reverse strategy can give a similar result by placing a FRT-flanked terminator cassette in front of the GAL4- coding sequences (Ito, 1997).

Another approach to regulate temporal expression of a transgene is to use a subtractive gene strategy. This strategy was first introduced as an essential component of the MARCM system for Mosaic Analysis with a Repressible Cell Marker, which combines Flp/FRT and Gal4/Gal80 to couple transgene expression with mitotic recombination (Lee, 1999). The GAL80 protein binds to the C-terminus of GAL4 and blocks transcription by interfering with the recruitment of other components of the transcriptional machinery. The use of GAL80 repressor offers extensive possibility to express a transgene in a restricted population of cells by using the logic gate in population expressing A NOT B.

4.2.3.     Split protein system

This strategy rests on components that are inactive alone, but when combined reconstitute a desired function as for protein complementation, in which two inactive fragments of a protein associate to reconstitute function. By independently targeting the expression of the two non-functional parts of a split protein, reconstitution of function can be achieved at the intersection, and only at the intersection, of the expression patterns of the two promoters used to target them. The typical example is the “split-GAL4” method that takes advantage of the modular nature of the GAL4 transcription factor (Luan, 2007). In this technique the separate DNA binding domain (DBD) and activation domain (AD) of GAL4 are fused to a heterodimerizing leucine zipper motif and each fusion protein is expressed separately. Only when they are present in the same cell can the leucine zippers direct heterodimerization, resulting in the formation of a functional activator (Figure 2E).

A similar strategy has been used to design a ‘‘split-Cre’’ system based on the complementation of Cre protein fragments. Here, the Cre recombinase was divided in two halves and both Cre fragments were fused to the constitutively dimerizing coiled-coil leucine zipper domain of the yeast transcriptional activator GCN4 to force the association of split-Cre fragments, thereby enhancing Cre activity by functional complementation (Hirrlinger, 2009b).

4.3.     Temporal control of transgene expression

While global expression of transgenes can be used as an effective tool for studying the consequences of gene activation or inactivation, in many instances it is desirable to control both spatial and temporal aspects of transgene expression. As described above, spatial resolution is achieved by the use of tissue- or cell-specific promoters but timing of transgene expression can be controlled by addition or withdrawal of a small molecule able to either induce or inhibit gene expression.

A widely used tool in both mouse and fly is the tetracycline-dependant expression of transgene. This system is based on the transcriptional activity of the tetracycline-transactivator (tTA). This transcription factor was modified by the incorporation of a fusion between the Escherichia coli tetracycline repressor and the strong transcriptional activation domain of the herpes simplex virus VP16 (TetR-VP16). TetR-VP16 is expressed under the control of a tissue-specific promoter and can promote expression of genes bearing tetO operator sequences. Addition of the cell-permeable ligand tetracycline or its derivatives (e.g., doxycycline), which binds to and prevents TetR-VP16 from binding tetO (Gossen, 1992), can be used to repress expression at specific times. Two versions of the tetracycline systems exist: Tet-On, in which the addition of the drug results in an active reverse-tetR (rtTA) causing transgene activation from the tet operator and Tet-Off, in which addition of the drug inactivates tTA, and in turn, expression from the tet operator is switched off (Figure 2F). To take advantage of the number of established tissue-specific GAL4 lines, both the Tet-On and Tet-Off expression systems have been linked to the GAL4/UAS system (Stebbins, 2001). To do this the tetracycline transactivators were placed under the control of the UAS. Therefore, expression of rTA can be regulated by crossing the UAS-rTA (or an optimized version of rtTA with a high level of transgene induction called rtTAs-M2-altTA) transgenics to a given GAL4 driver strain (Stebbins, 2001).

Another similar approach to regulate temporal expression is to use hormone inducible variants of GAL4. Two systems are available, GAL4-estrogen receptor (GAL4-ER) (Han, 2000) and a second called GeneSwitch, which is a fusion of GAL4-progesterone receptor and the activation domain of p65 (Osterwalder, 2001) (Figure 2G). In the GAL4-ER, the receptor is engineered such that exogenous administration of the appropriate ligand results in the fusion protein relocating into the nucleus to activate transcription upon the administration of tamoxifen, an estrogen analog. Similarly, Cre recombinase fused to the estrogen receptor (Cre-ER), and expressed in specific tissues, can be temporally activated by the addition of tamoxifen (Feil, 1996) (Figure 2H), Drawbacks include the relatively slow response in gene expression following the cessation of therapy and the difficulties of delivering an oil-soluble ligand. It led to the development of a second generation mutant Cre-ER(T2), which gives a 4-fold increase in the efficiency of recombination induced by 4-hydroxy-tamoxifen in cultured cells (Indra, 1999). A refinement of this strategy called split-CreERT2 has been recently developed to allow spatially and temporally precise genetic access to cell populations defined by the simultaneous activity of two promoters (Hirrlinger, 2009a).

Proteins whose functions are regulated by temperature give an alternative strategy to small molecules for temporal control. For example, in Drosophila, a heat-shock-promoter driver Flp recombinase (hsFlp) has been developed to induce gene expression in a temporally controlled manner. Another example is the development of the temporal and regional gene expression targeting (TARGET) technique (McGuire, 2003). In this approach a temperature-sensitive variant of the GAL80 protein (GAL80ts) is expressed ubiquitously under the control of the tubulin 1α promoter. GAL80 repression of GAL4 is activated by a simple temperature shift (18 to 35°C), giving a precise temporal control of the onset of expression. Interestingly, the TARGET system is fully compatible with the vast array of GAL4 lines already established (Figure 2I).


5.     Viral mediated gene delivery

         Classical methods of gene transfer, such as transfection, have many limitations for gene delivery. First they are only applicable to cell cultures in vitro and then are often limited to dividing cells. The development of viral vectors has circumvented these limitations. Replacing genes necessary for viral replication with an expression cassette containing the genes of interest transforms the viruses into safe vectors able to deliver genetic material to various tissues including the brain. Viral vectors can also be combined with transgenic animals and/or genetic strategies described in chapter 3. In this section, we will describe viral vectors currently used for gene transfer into the brain and recent developments in viral technology to improve targeting, transcriptional regulation and transgene expression.

5.1.     Viral vector description

Viral expression systems combine many advantages including fast and easy implementation, high expression of transgenes in infected cells and efficiency in a lot of species such as primates where transgenesis is difficult to perform. Here, we will present briefly several kinds of viruses, including retrovirus, adenovirus, adeno-associated virus and herpes simplex virus that are used in gene transfer into the brain (Table 1).

5.1.1.     Retrovirus/Lentivirus vectors

Retroviruses are a class of enveloped single stranded RNA virus. Following infection, the viral genome is reverse transcribed into double stranded DNA, which integrates into the host genome and is expressed as proteins. The viral genome is about 7-10kb, composed of three gene regions termed gag (coding for viral protease and integrase), pol (coding for reverse transcriptase) and env (coding for the viral envelope glycoprotein). At each end of the genome are long terminal repeats (LTRs), which include promoter/enhancer regions and sequences involved in integration. The genome also has a packaging signal (Y) and RNA splice sites in the env gene. Retroviral vectors are most frequently based upon the Moloney murine leukaemia virus (Mo-MLV), an amphotrophic virus, capable of infecting both mouse cells (via the cationic amino acid transporter CAT-1 (Weiss, 1995)) and human cells (via the transmembrane phosphate transporter RAM-1 (Weiss, 1995)).

In the recombinant retroviral vector, the viral genes are replaced with the transgene of interest and expressed on plasmids in the packaging cell line. Because the non-essential genes lack the packaging sequence (Y) they are not included in the virion particle. To prevent recombination resulting in replication competent retroviruses, all regions of homology with the vector backbone should be removed and the non-essential genes should be expressed by at least two transcriptional units (Markowitz, 1988). Even so, replication competent retroviruses do occur at a low frequency. With this system, it is possible to produce viral titres of 105-107 colony forming units/ml.

The disadvantages of retroviral vectors include the random insertion into the host genome, which could possibly cause oncogene activation and the limited insert capacity (around 8kb). Moreover, a requirement for retroviral integration and expression of viral genes is that the target cells should be dividing. This limits its use to proliferating cells in vivo. For example, when treating cancers in vivo, tumour cells are preferentially targeted (Roth, 1996).

Lentiviruses belong to the general category of retroviruses that are mostly based on the human immunodeficiency virus type 1 (HIV-1). Once inside cells, the RNA genome of the lentivirus is reverse transcribed by reverse transcriptase into a double-stranded DNA provirus that is incorporated into a pre-integration nucleoprotein complex able to pass through the pores of intact nuclear membranes. Therefore lentiviral vectors have all the advantages of Mo-MLV-based vectors, alongside the ability to infect both dividing and non-dividing cells (Naldini, 1996). HIV vectors can accommodate fairly large gene inserts and can provide long-term expression through chromosomal integration. Lenti-vectors have a capacity of ~10 kb for genetic material and sufficient amounts of high-concentration vector (108 to 109 infection units/mL) can easily be produced. Stability of expression of lentivirus vectors in the brain is their greatest advantage. Long-term expression of the transgenes has been observed in rat neurons for at least 6 months following intracerebral injection of lentiviral vectors, with no sign of tissue pathology or immune response (Blomer, 1997). Nevertheless, a major limitation of lentiviruses is the limited genetic payload length that is often incompatible will full size enhancer/promoter needed for strong and cell-type specific expression.

5.1.2.     Adenovirus vectors

Adenoviruses are large (60-90nm diameter), non-enveloped, linear double-stranded DNA viruses that are usually associated with mild human infections including upper respiratory tract infections, keratoconjunctivis and gastroenteritis. The adenovirus genome is 36kb in length and contain inverted terminal repeat (ITR) sequences at both ends containing the cis-acting DNA sequences that which define the origin of DNA replication. The gene transcription of this virus can be divided into two phases of gene expression: early genes (E) and late gene (L), expressed before and after the onset of viral DNA respectively. The first mRNA/protein to be made around 1h after infection is E1A. This protein is a trans-acting transcriptional regulatory factor that is necessary for transcriptional activation of early genes. The protein is also capable of activating transcription from a variety of other viral and cellular promoters and shows no sequence-specificity, indicating that it functions by modifying the cellular environment.

The uptake of the adenovirus particle is a two stage process involving an initial interaction of the fibre knob protein with a range of cellular receptors, which include the MHC class I molecule and the high affinity cell surface receptor called the Coxsackie and adenovirus receptor (CAR) (Bergelson, 1997). The capsid penton base protein then binds to the avb3 and avb5 integrin family of cell surface heterodimers allowing internalization via receptor-mediated endocytosis (Wickham, 1993). Most cells express primary receptors for the adenovirus fibre coat protein, however internalisation is more selective.

The first generation of recombinant adenovirus (rAds) is derived from the human adenovirus serotypes 5 and 2 and their replication was made defective-through deletion of the E1 gene regions. Adenoviruses are commonly used for gene transfer, as they can be generated at high titres and efficiently infect and express their genes in a variety of cell types including both dividing and quiescent cells. Other advantages of this vector include ease of manipulation (Graham, 1991) and large insert capacity up to 35 kb of foreign DNA (Schiedner, 1998).

This vector has some drawbacks that may prevent its future use. First, most adenoviral vectors in their current form are episomal thus they do not integrate into the host DNA and therefore only cause a transient transgene expression. Second, because most individuals have been exposed to natural adenovirus infections, immunologic responses may hamper gene transfer efficacy. In addition, because the adenoviral genes express hundreds of proteins adenoviruses stimulate the immune system and trigger inflammatory responses. Newer second- and third-generation of rAd vectors that are deficient or defective in the E2, E3 or E4 gene regions are less immunogenic than the first generation and can be propagated on trans-complementary cell lines (Brough, 1996).

Vector systems have been developed in which most or all adenovirus proteins coding sequences are removed. From the viewpoint of safety, size of transgene and absence of vector gene expression, these so-called 'gutless' adenovirus vectors contains only the ITRs and packaging signal of the wild type virus. The gutless vectors require virtually all adenovirus gene functions to be provided for vector propagation and, since this cannot yet be achieved using a packaging cell line, they must be provided using a helper cell (Parks, 1996). Interestingly, gutless adenoviruses can be produced with a high titre (Parks, 1996) and give rise to long-term expression compared with first generation adenoviruses (Morsy, 1998).

5.1.3.     Adeno-associated vectors

Adeno-associated virus (AAV) is a small (25 nm diameter), non-enveloped and single-stranded DNA parvovirus. The upstream open reading frame encodes four replication (rep) proteins that allow AAV rep-proteins to package AAV ITR–flanked transgenes into nearly all serotype virions. As a dependovirus, AAV requires Adenovirus or Herpes Simplex Virus (HSV) as a helper virus to complete its lytic life (Conway, 1997). In the absence of the helper virus, wild type AAV establishes latency by integration with the assistance of Rep proteins through the interaction of the ITR with chromosome 19 (Berns, 1995). The first stage in the AAV life cycle is the binding of the particle to a host cell. To bind cell receptors, AAVs use heparan sulphate proteoglycan (HSP) structures on the cell surface. These structures are com of two or three HS groups located near the cell surface or extra-cellular matrix. Once bound to these receptors, they utilize co-receptors on the cell surface, for example, avb5 integrin and fibroblast growth factor receptor 1 (FGFR1), which aid in internalization via receptor-mediated endocytosis followed by endosomal sorting and trafficking into the nucleus (Ding, 2005).

Until recently, the majority of the research conducted using AAV-based vectors employed serotype 2. Vectors based on AAV2 have been the most studied and are currently used in clinical trials for some diseases (Hildinger, 2004). To date at least 10 additional serotypes of AAV have been identified, the majority of which have been isolated as contaminants of adenoviral cultures. Many in vivo studies have clearly demonstrated that the various AAV serotypes display different tissue or cell tropisms (Zincarelli, 2008).

Recombinant AAV (rAAV) vectors are constructed by co-transfection of two plasmids. The first one contains the transcription unit of interest flanked by the ITRs and the second contains the rep and cap ORFs. In order to propagate rAAVs, infection with a helper virus (classically an adenovirus) is required. Although this technique enables the production of rAAVs with high titres (1010 infectious particles/ml) (Samulski, 1989), it requires extensive purification steps involving heating and caesium chloride gradient purification to remove adenoviral contamination. Here again, a main drawback is the packaging capacity for the transgene (4,7kb) that is too limited to generate vectors with a restricted expression in a specific cell type. Nevertheless, AAV has gained attention because of its safety, lack of immunogenic viral proteins and efficient transgene expression in a very broad host range (Samulski, 1989). AAV is able to infect a large number of both dividing and non-dividing cells including neurons (Miao, 2000). AAV is not pathogenic and is not associated with disease, even though it has a broad range of infectivity. Moreover, transgene expression from rAAV vectors has been shown to continue for long periods of time, including up to 15 months in the CNS (Lo, 1999).

5.1.4.     Herpes simplex virus vectors

Herpes simplex virus (HSV) is an enveloped, doubled-stranded DNA virus with a genome of around 150 kb encoding at least 80 proteins including many enzymes and surface glycoproteins. HSV is a member of the Herpesviridae family. It possesses an icosahedral capsid and is considered to be relatively large for a virus, with virions ranging from 120 nm (nanometres) to 300 nm in size. HSV is a neurotropic DNA virus with a wide host range due to binding of viral envelope glycoproteins (gB ang gC) to the extracellular HSP molecules (WuDunn, 1989). Internalisation of the virus requires FGFR1 and envelope glycoprotein gD that binds specifically to a receptor called the Herpes Virus Entry Mediator receptor (HVEM) and provides a strong, fixed attachment to the host cell (Kaner, 1990).

Three different classes of vectors can be derived from HSV: replication-competent attenuated vectors, replication-incompetent recombinant vectors and defective helper-dependent vectors known as amplicons (Neve, 2005). Amplicons are HSV-1 particles identical to wild-type HSV-1 from the structural, immunological and host-range points of view, but which carry a concatemeric form of a DNA plasmid, named the amplicon plasmid, instead of the viral genome. An amplicon plasmid is a standard E. coli plasmid carrying an origin of DNA replication and a cleavage/packaging signal (pac or “a” site) from HSV-1, in addition to the transgenic sequences of interest. The major interest of amplicons as gene transfer tools stems from the fact that they carry no virus genes and consequently do not induce synthesis of virus proteins. Therefore, these vectors are fully nontoxic for the infected cells and non pathogenic for the inoculated organisms. A second and major advantage is that most of the 150 kbp capacity of the HSV-1 particle can be used to accommodate very large pieces of foreign DNA such as BAC clones allowing integration of full size enhancer/promoter for cell-specific expression. Herpes viruses are currently used as gene transfer vectors due to their specific advantages over other viral vectors. Among the unique features of HSV derived vectors is their ability to invade and establish lifelong non-toxic latent infections in neurons (Carpenter, 1996).

5.2.     Targeted gene delivery

Viral vectors present many advantages for specific cell type delivery of transgene compared to transgenesis and DNA electroporation. In fact, recent developments have focused on the improvement of cell-type specificity in brain by using modified viral tropism, specific promoters and intersections of viral infection with genetically modified organisms.

5.2.1.     Modification of viral tropism

At the cellular level, a virus undergoes 5 major steps prior to achieving gene expression: 1) binding or attachment to cellular surface receptors, 2) endocytosis, 3) trafficking to the nucleus, 4) uncoating of the virus to release the genome and 5) conversion of the genome to double-stranded DNA as a template for transcription in the nucleus. In this cascade of events, the first step, often termed viral tropism, defines the specificity of a virus for a particular host cell type. Therefore by replacing the envelope or capsid proteins from a virus with that of another virus, the host range can be extended, in a technique known as pseudotyping.


The retroviral envelope interacts with a specific cellular protein to determine the target cell range. Altering the env gene or its product has proved a successful means of manipulating the cell range. Approaches have included direct modifications of the binding site between the envelope protein and the cellular receptor, however these approaches tend to interfere with subsequent internalisation of the viral particle (Harris, 1996). Another strategy to target specific cell types is to genetically insert an antibody bridge between the envelope glycoprotein and specific cellular receptors (Etienne-Julan, 1992). Nevertheless, the retroviral vector constructed from the murine leukemia virus can only express transgenes in cells undergoing mitosis, indicating its inability as a delivery vehicle for neuronal expression.

Recombinant lentiviruses are typically modified with the VSV-G glycoproteins, mainly because they allow easy concentration to high titres by ultracentrifugation. At the same time, because the VSV-G glycoproteins bind to ubiquitous phospholipid components of the plasma membrane but not to a specific cell surface receptor, such viruses have an extremely broad host-cell range (Burns, 1994). Thus, lentiviral expression of transgene in the brain is determined by selection of a neuronal specific promoter rather than specific tropism (see chapter 5.2.2.).


Targeting cell entry of adenoviruses requires both the ablation of the native adenovirus tropism, especially for hepatocytes, and the incorporation of targeting ligands into the virus capsid. These aims have been achieved by complexing adenoviruses with different types of bispecific adapter molecules able to bind to specific cell-surface receptor (Barnett, 2002). To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with monoclonal antibodies directed against surface-expressed target antigens and more (for review, see (Coughlan, 2010)).

Adeno-associated virus

AAV serotype 2 (AAV2) was the first AAV serotype to be cloned into bacterial plasmids (Samulski, 1982). Since its discovery, 10 other serotypes with different capsid proteins affecting receptor-mediated endocytosis of AAV particles have been characterized (Rutledge, 1998). The tropism of AAV has been limited to particular cell types but can be expanded to include other cell types through modification of the capsid to target specific cells or enhance AAV transduction. Thanks to the high degree of homology between the amino-acid sequences of the different AAV serotypes and knowledge about the AAV2 crystal structure, it is possible to form a virion shell from capsid subunits of different serotypes to generate AAV that are composed of a mixture of viral capsid proteins from different serotypes. These mosaic virions exhibit a broader tissue tropism due to the combination of the tropisms from different serotypes, and also exhibit enhanced transgene expression since different serotypes may have different cellular trafficking pathways that serve to initiate transgene expression more efficiently (Cearley, 2006, Gao, 2005, Taymans, 2007). A recent study of neuronal infectivity for different serotypes has demonstrated that different brain regions exhibit different patterns of transduction (Taymans, 2007).

As for retroviruses, AAV tropism can be expanded to include other cell types by modification of receptor targeting by using an AAV-specific antibody that is chemically linked to another antibody binding specifically to a cellular receptor known to be expressed on the targeted cell surface. An example is the pioneering experiment performed with AAV2 able to recognize megakaryocyte cells (Ponnazhagan, 1996). Another strategy is the genetic manipulation of the capsid gene by insertion of a foreign protein sequence either from another wild type AAV or an unrelated protein (for review see (Choi, 2005)). A good example is the expression of the cellular receptor for retroviral envelope protein by an adeno-associated vector expressing improved viral transduction into numerous cell lines (Qing, 1997). Finally, it is possible to enhance AAV transduction by transcapsidation that consists in packaging an AAV genome containing an ITR from one serotype into the capsid of another serotype (Rabinowitz, 2002).

Herpes simplex virus

HSV enter their host via mucosal epithelia, skin or cornea. While humans are the only natural host, HSV can infect a wide range of mammalian species under laboratory conditions, indicating a broad host range. Because HSV has a very broad host range, the utility of HSV vectors may be greatly increased by restricting viral infection to the cell types of interest, particularly in applications in which virus replication is required for effective gene delivery. (for review, see (Coughlan, 2010)). To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping capsid proteins derived from other viral families, monoclonal antibodies directed against surface-expressed target antigens and other strategies (for review, see (Coughlan, 2010)).


5.2.2.     Transcriptional targeting

One of the major challenges in targeted gene transfer is the specificity of transgene expression only in the cell types of interest. As complex mechanisms regulate gene expression in vivo and most viral promoters do not have specific targeting capacities, a variety of tissue or cell specific promoters have been characterized. Moreover, viral promoters such as human cytomegalovirus immediate-early gene (CMV) are commonly used as regulatory element due to their strong activity in various cell types in vitro, but they are often not suitable for long-term expression in neurons. As discussed in previous chapters, promoters in mammals are several fold larger than their viral counterparts (up to hundred of kb), which is incompatible with the packaging capacity of all viral vectors except for the last generation HSV amplicons. To overcome this limitation, hybrid and synthetic promoters are being developed in order to improve the cell-type specificity and provide high level of transgene expression. A recent study, it was reported on the ability of short promoter sequences to drive fluorescent protein expression in specific types of mammalian cortical inhibitory neurons using adeno-associated virus and lentivirus vectors (Nathanson, 2009a). This group demonstrated that among fugu compact promoters PV, CR, SST and NPY only the somatostatin and the neuropeptide promoters largely restricted expression to GABAergic neurons. Moreover, GFP expressing lentivirus vector that can accommodate larger regions of these promoters drove expression in excitatory neurons but not in inhibitory neurons consistent with the expected differences due to viral tropism (Nathanson, 2009b).

These results highlight the complexity of gene regulation and our lack of knowledge about regulatory elements that are required to precisely restrict expression in neurons. Nevertheless, many neuron specific promoters have been used for transcriptional targeting by viral vectors, including those that control the expression of genes encoding neuron specific enolase (NSE), synapsin-1 (SYN), platelet-derived growth factor (PDGF), tyrosine hydroxylase (TH), and dopamine β-hydroxylase (DBH) (Fitzsimons, 2002, Glover, 2002, Kugler, 2001, Paterna, 2002).

5.2.3.     Intersectional genetic switches

Intersectional strategies described in detail in chapter 3 are currently the most efficient approaches to achieve high cell-type specificity and have therefore been adapted for use with viral vectors. For example, a highly specific neuronal expression in rat brain was achieved using adenoviral infection (Namikawa, 2006). By combining an adenovirus expressing Cre recombinase under the control of a modified promoter of the superior cervical ganglion10 (SCG10) with another adenovirus vector expressing a Cre inducible EGFP flanked by loxP sites, this group was capable of mediating transgene expression at high levels both in neuronal cells of mixed cultures and in an animal model. The Cre/lox system has also been used as a random gene splicing strategy to express various combinations of fluorescent proteins in individual neurons of the brain of a transgenic mouse called brainbow mouse (Livet, 2007). More recently, this approach has been adapted for use with rAAV to deliver opsin gene in defined cell-types (Figure 3A). The specificity of this system is very high and relies on the introduction in the virial backbone of an ubiquitous promoter such as translational elongation factor EF1a in front of the gene encoding ChR2-YFP surrounded by two pairs of heterotypic and antiparallel loxP/lox2272 recombination sites (Atasoy, 2008, Kuhlman, 2008, Sohal, 2009). When this rAAV is stereotaxically injected in the brain of a Cre transgenic mouse (Figure 3B), only neurons selectively expressing of Cre are able to process the cassette and therefore will express of the ChR2-YFP carried by the viral construct (Figure 3C). Another advantage of this strategy is the low false positive background due to transcriptional read-through observed with classic lox-STOP-lox cassette (Kuhlman, 2008).

In addition to achieving cell-type specific expression, it would be desirable to achieve regulation of transgene expression. Here again, several regulatory presented in chapter 3 have currently been adapted for use with viral vectors. For example, the tet system has been shown to be functional when expressed from several viral vector enabling tight regulation and inductility of transgene expression (Fotaki, 1997, Harding, 1998, Hwang, 1996).

5.3.     Circuit mapping

The ability to visualize complex and extended neural networks is critical to understanding the functional organization of the brain. Classically, brain circuit mapping has been established with chemical probes but this method lack cell type specificity. To overcome this problem, a useful strategy uses viral tropism to target cell types based on their axonal projections. For example, different types of cortical pyramidal neurons project axons to distinct distant targets. Viruses that can efficiently infect neurons through their axon terminals can therefore be injected into a particular target structure, resulting in the selective infection of neurons that have axons in that structure. This method has been successfully employed using HSV amplicon vectors, recombinant rabies virus, and adenovirus, as well as lentivirus pseudotyped with the rabies virus envelope protein (Mazarakis, 2001, Sandler, 2002, Tomioka, 2006, Wickersham, 2007). For example, adenovirus vectors can be transported in a retrograde manner from the injection site to the projection cell bodies, following uptake at nerve terminals (Akli, 1993, Ridoux, 1994). Another interesting application of this ability of adenovirus to be retrogradely transported is that it could be used for the specific targeting of selected neuronal populations not easily accessible by direct injection, while avoiding any undesirable side effects associated with systemic administration (Finiels, 1995) or tissue damage due to viral toxicity at the site of injection (Cayouette, 1996). Trans-synaptic targeting may also be achieved by expressing genetically encoded neuronal tracers such as wheat germ agglutinin (WGA) (Gradinaru, 2010) or tetanus toxin C (TTC) (Schwab, 1979). Recently, monosynaptic inputs of specific neuronal cell types have been determined by injection of glycoprotein-deleted (DG) rabies virus in transgenic mice that conditionally express rabies glycoprotein (Weible, 2010). Moreover, 12 new DG rabies virus variants were recently developed including ChR2-mCherry to allow selective light-controlled neuronal activation on the basis of their connectivity (Osakada, 2011).

6.     Conclusion

         A strategy to answer the difficult question of how the brain works is:  to know, to modify and to control neuronal activity. To know means to identify the meaningful genetic markers that are expressed in the cell-types of interest within the brain. Even if this work is a long-drawn-out job, our knowledge of enhancers and promoters controlling gene expression in the CNS will be greatly improved by international projects on whole transcriptome or protein expression at single cell level. This will give the cis-regulating elements and/or the specific cell surface receptors that can be targeted by a virus, depending on the strategy to express a transgene in a chosen population of neurons. Based on this dataset, to modify relies on the technical ability to access the genome of these selected cells. Transgenic technologies have emerged as invaluable tools to manipulate the genes in neuroscience research. However, this method is still hampered by a relatively low efficiency and high cost in species other than mice. For this reason, the use of viral mediated gene delivery paves the way for an easier, faster and cheaper modification of host genome and on a wider range of species. Then, controlling neurons will become possible thanks to optogenetics by using the most suited strategy to express opsins in the cells of interest with the greatest strength and specificity. When the first two steps will be fully mastered, optogenetic technology will extend even more our capacity to control a variety of neuronal networks with the highest possible spatiotemporal resolution, which is necessary to go further in the understanding of complex processes that occur in the brain.



Figures and Table Legend


Table 1 Properties of viral vectors used for gene delivery in the brain.


Figure 1 Methods for targeting gene expression


A) Bacterial artificial chromosomes (BACs) are large-insert DNA clones that can accommodate up to 200 kb of genomic DNA, and are likely to contain all the regulatory elements E/P necessary to confer accurate transgene expression in vivo.

B) The enhancer trap technique uses a reporter gene fused to a minimal promoter, typically containing the TATA box and transcription start site. The minimal promoter is located on the reporter gene so that it can only be activated by nearby enhancer sequences near the chromosome insertion site.

C) Gene knock-in refers to a genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in the genome.

D) Transgenes targeted by homologous recombination to the Rosa26/Hipp11 locus are stably and efficiently expressed in undifferentiated cells as well as the differentiated cell types generated from mouse ES cells.

E) Plasmid derived from bacteriophage FC31 inserts its target gene into that of its host via the integrase (INT) enzyme, which catalyzes recombination between a phage attachment site (attP) and a attachment site (attB) present in the chromosome. Integrase requires no accessory factors and has a high efficiency of recombination.


Figure 2 Genetic strategies for refined gene expression

A) The GAL4 protein is present only where the promoter A (E/PA) is active. GAL4 binds to a sequence called the upstream activating sequences (UAS) element and is induces translation of the gene linked to UAS

B) Cre/loxP or Flp/FRT leads to expression in cells where the promoter A is active and therefore removes the transcription stop to allow expression of the target gene under the control of a constitutive promoter P. LoxP and FRT consists of a 34 bp DNA sequence containing an asymmetric 8 bp sequence (red) in between two sets of palindromic, 13 bp sequences flanking it.

C) A combination of Cre/loxP and Flp/FRT recombination systems allows for the target gene to be expressed only when promoters A and B are active in the same cell.

D) As for C, a combination of GAL4/UAS and Flp/FRT increases cell specificity for the target gene which is only expressed when promoters A and B are active.

E) Split-GAL4 system, which independently targets the Gal4 DNA-binding domain (Gal4DBD) and a cognate transcription activation domain (AD) using two different promoters A and B, drives transgene expression in a restricted fashion: only cells in which both promoters are active at the same time express the two heterodimerizing transcription factor domains to reconstitute transcriptional activity. This system is also available for Cre recombinase.

F) Tetracycline inducible GAL4 system encompasses two complementary control circuits, described as tTA dependant (Tet-Off) or rtTA dependant (Tet-On) expression system. Expression of the target gene is only possible in cells where promoter A is active and if doxycycline (Dox), a tetracycline analog is present (Tet-on) or absent (Tet-Off).

G and H) Inducible version of GAL4-ER/UAS and Cre-ER/loxP recombinase systems in which addition of tamoxifen will cause dose-dependent activation of the target gene. ER: estrogen receptor.

I) Temporal control of the GAL4/UAS system is based on a temperature sensitive GAL80ts able to repress expression of the target gene at 18°c but not at 29°C.


Figure 3 Highly specific viral mediated gene delivery

A) Schematic representation of DIO/FLEX system to express ChR2-YFP in a specific neuronal subpopulation. This strategy is based on two components: a Cre dependant virus containing ChR2 expression cassette under the control of a ubiquitous promoter P and a Cre driver transgenic mouse expressing Cre recombinase in a specific population of cells. After viral infection, the transgene integrates into the host genome facilitating stable expression of ChR2-YFP.

B) Experimental setup used to inject viral particles in mouse brain. 1) A stereotaxic apparatus is used to inject virions in the region of interest with high precision (+/-10 µm) in an anesthetized mouse. 2) Body temperature is maintained around 37°C during surgery on a heat-controlled blanket. 3) For viral injection, thin holes are drilled through the skull (top right panel) under the guidance of a stereomicroscope. 4) After the micropipette is positioned in the brain parenchyma at the desired coordinates, the virus is injected with a constant speed. 5) The speed and volume of the injection is monitored by injector system (Stoelting QSI). The bottom right panel shows typical fluorescence observed after 14 days of infection by using rAAV2/1-FLEX-ChR2 in PV-Cre transgenic mouse (Hippenmeyer, 2005) (image is a composite of bright field and YFP epi-fluorescence images). White dot: bregma position, white triangle: lambda position, LH: left hemisphere, RH: right hemisphere. Unpublished results.

C) The right panel shows a high-resolution mosaic image in the YFP channel (consisting of around 100 individual frames) obtained through an automated Zeiss microscope, equipped with a high-precision motorized stage. The mosaic image was obtained from entire coronal brain sections cut at 40µm thickness. Level of ChR2 expression in somatosensory cortex was assessed by YFP visualization. Control Alexa568-labelled anti-parvalbumin antibody (Swant) show correct immunostaining of PV expressing interneurons (white triangle) under confoncal microscopy (bottom right panel). The top right panel indicates coordinates used for virus injection and a schematic representation of the injection site in the mouse brain (red star).  Ppac: posterior parietal association cortex, Ssbf: somatosensory barrelfield, Aud: Auditory cortex, CTX: cortex, HPC: hippocampus, TH: thalamus. Urban A et al. Unpublished results.


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