Transgene array inserted into chromosome
Transgene array inserted into chromosome been mutated, to explore the effect of expressing a mutant protein in vivo, or to produce an animal model with a very specific phenotype (such as epilepsy). When considering a transgenic approach to developing a mouse epilepsy model, three questions must be addressed: (1) What sort of transgene would cause a mouse to have seizures? (2) Where in the mouse would it need to be expressed? (3) When during the animal's life should it be expressed? Because there are so many different possible transgenes (and a similarly wide range of mechanisms) that could cause seizures or epilepsy in a mouse, the choice of transgene depends on the specific needs or interests of the investigator. In this chapter, we discuss primarily ion channel genes.
Two general categories of ion channel genes can be used to create a transgenic mouse model of epilepsy: wild-type and mutant. A wild-type transgene can be expressed in the same cell type as the endogenous version of that gene if the appropriate promoter is available, or it can be expressed in a cell type that does not normally express the endogenous gene. It might seem intuitive that overexpressing a gene that increases membrane excitability (e.g., a voltage-gated Na+ channel) would favor the development of seizures and epilepsy, whereas overexpressing a gene that decreases membrane excitability (e.g. a K+ channel) would inhibit seizures. However, this scenario is not always realized. In some cases the genome can employ feedback regulation to alter the expression of endogenous genes to compensate for the effects of a transgene. Transgenic expression of a mutant ion channel gene can also be used to create an epilepsy model, but not all ion channel mutations associated with human epilepsy are good candidates for this purpose. Because a transgenic mouse already has two functioning endogenous copies of every gene, only those mutant ion channel transgenes that carry gain-of-function mutations (not those that carry loss-of-function mutations) might be expected to replicate a human seizure phenotype.
The technique of transgenesis via microinjection into mouse embryos was described more than 25 years ago (Gordon et al., 1980). An overview of that process is shown in Figure 2. Most transgenes consist of the same elements as endogenous genes and include a promoter, a region of transcribed sequences that encode a protein, and a poly(A)-addition signal. The protein coding segment usually consists of an intronless cDNA fragment rather than a fragment of genomic DNA containing the gene because the latter tends to be too large for simple cloning procedures (notable exceptions include transgenes consisting of entire bacterial artificial chromosomes, or BACs). A transgene is constructed by standard recombinant DNA techniques in a plasmid vector and is amplified by growth in Escherichia coli. The vector portion (which can interfere with transgene expression) is
FIGURE 2 Construction of a transgenic mouse model. A typical transgene plasmid contains a promoter, structural gene, and poly(A) addition signal. This is amplified in Escherichia coli (1), linearized to remove vector sequences (2), and purified. The purified transgene fragment is micro-injected into one pronucleus of a recently fertilized mouse zygote (3). The transgene usually inserts as a multicopy tandem array at a single site in the genome (bottom). The transgenic zygote is implanted into a surrogate female (4) to develop until birth about 20 days later.
removed, and the purified transgene fragment is diluted in a physiologically buffered solution. This transgene-containing solution is then microinjected into one of the two pronuclei of a newly fertilized zygote that has been surgically removed from a recently mated female donor mouse (the female is usually treated with hormones before mating to cause "superovulation," the production of a greater number of mature ova than generated during a normal cycle). Following fertilization, the haploid egg and sperm pronuclei do not fuse immediately. Because the male pronucleus is usually larger than the female pronucleus, it is generally an easier target for the microinjection needle. Thus, most transgenes actually insert into the paternal genome. It is also interesting to note that certain mouse strains produce zygotes with larger pronuclei than others or that can survive the process of microinjection better and therefore are technically easier to access. These practical concerns are often balanced by a desire to create the transgenic mouse on a specific background strain.
Following microinjection, multiple copies of the transgene concatemerize (head-to-tail, head-to-head, tail-to-tail, or a combination of these) and insert into a single site in the genome. More rarely multiple transgene arrays insert into different sites in the genome. It remains unclear whether the resulting copy number in the transgene array is correlated with the concentration of transgene DNA in the microinjection solution. The insertion site in the genome is believed to be essentially random. In fact, 3 to 5% of transgenes insert into an endogenous gene, resulting in an insertional mutation that can confound analysis of the transgenic mouse phe-notype (Meisler, 1992). The microinjected embryos are then implanted into a pseudopregnant surrogate female that had been previously treated with hormones to facilitate implantation. The mice that are born about 20 days later (referred to as "founders," or F0-generation pups) may or may not carry the transgene; they must be screened, for example, by carrying out polymerase chain reaction (PCR) genotyping of genomic DNA extracted from a blood sample, a tail clipping, or an ear-punch tissue sample. An alternate approach to creating transgenic mice is to infect mouse embryos with a retrovirus engineered to express a transgene, which then inserts itself into the genome using natural viral mechanisms (Ivics and Izsvak, 2004). This approach is used less frequently than embryo microinjection. However, because retroviral transgenes tend to insert as a single unit rather than as a variably sized tandem array, this approach may provide the investigator with some control over transgene copy number. It is unclear whether retroviral transgenes insert at random into the target genome or preferentially at certain sites (Wu and Burgess, 2004).
Although a basic transgene contains a promoter, a protein-coding gene segment, and a poly(A)-addition signal, there are variations that can be useful (Figure 3). Some studies have shown that inclusion of a single intron in the transgene can increase expression significantly, perhaps by increasing pre-mRNA stability or mRNA nuclear export. This intron does not need to be derived from the same gene that is being expressed and is usually positioned between the promoter and the translation start site (first ATG codon) of the transcribed gene. Another variation is the addition of chromatin scaffold attachment regions, or SARs (also known as matrix attachment regions, MARs, or insulators). These are fragments of DNA that are thought to regulate local chromatin structure (Bode et al., 2003; McKnight et
Promoter Gene Poly(A)
SARs added: SAR Promoter Gene Poly(A) > SAR
Epitope added: Promoter Gene Epitope tag Poly(A)
IRES vector: Promoter Gene 1 IRES Gene 2 Poly(A)
BAC transgene: Native PoyA)
FIGURE 3 Alternative transgene designs. Modifications (gray boxes) to the basic transgene design include the addition of introns to increase mRNA stability and nuclear export, chromatin scaffold attachment regions (SARs) to insulate the transgene from position-dependent effects on transcription, epitope tags (including GFP or LacZ) for identification or localization in vivo and an internal ribosome entry site (IRES) to allow the translation of two independent proteins from a single mRNA molecule. The bottom panel indicates that an entire bacterial artificial chromosomes (~80-300 kb) can be used as a transgene, with no additional modification, to preserve native regulatory features that might be lost when using cDNAs for transgenes.
al., 1992). By insulating the transgene and its promoter from the DNA environment into which the transgene inserts, flanking SARs can help to ensure that the transgene is expressed in the intended tissue and not influenced by nearby endogenous enhancer or repressor elements. This process can improve concordance between different mouse lines carrying the same transgene. Transgenes may also incorporate small (£20 amino acids) epitope tags at the n-terminus, c-terminus, or inside the transgene. These tags are translated as an integral part of the transgene protein and allow the use of existing antibodies to the epitope tag (myc, HA, and His tags are popular) to detect or immunoprecipi-tate the protein. Larger proteins can also be fused directly to the target gene, such as green fluorescent protein (GFP) or b-galactosidase (LacZ), which allows visualization of transgene expression without antibodies. However, the addition of these larger proteins may interfere with the functions of the gene under study. Finally some transgene vectors allow the simultaneous expression of two genes separated by an internal ribosome entry site (IRES). With this system, a single mRNA is transcribed, but then two separate proteins are translated from that transcript. This process can be useful for expressing a protein with a separate visualization label.
The choice of genes that can be expressed transgenically is practically unlimited. Currently the major constraint on transgene design is the availability of well-characterized promoters to drive tissue- or developmental stage-specific transgene expression. Promoters can be cloned from endogenous genes in an attempt to express the transgene in its native pattern; alternatively, promoters can be obtained from viral, bacterial, or even plant genes to drive expression in the mouse. With regard to expression in the central nervous system (CNS), a review of the literature reveals transgenes that incorporate Purkinje or granule neuron-specific, GABAergic neuron-specific, hippocampal, or pan-neu-ronal promotors. However, for a large number of interesting and functionally discrete brain regions or cell types, there are no currently available transgene promoters. It is extremely time consuming to clone and characterize new gene promoters, and some promoters are simply too complex or large to incorporate into a standard transgene construct. An increasingly popular approach is to express a gene from its native promoter via a BAC transgene (Gong et al., 2003; Heintz, 2000). The large size of most BACs (80-300kb) increases the likelihood that the complete promoter, intronic, and 3' regulatory sequences will also be contained within that clone. Unlike a standard transgene construct, a BAC does not need to be linearized or separated from its relatively small vector segments, but it can simply be purified, resuspended in the appropriate buffer, and directly microinjected into the zygote pronucleus. The technology to modify genes within a BAC clone (e.g., to introduce epitope tags, mutations, or other complex alterations)
is rapidly advancing (Giraldo and Montoliu, 2001; Testa et al., 2004).
In addition to autonomous promoters that either direct constitutive transgene expression or direct transgene expression in response to developmental cascades or natural cues, significant progress has been made toward developing transgene promoters that are truly inducible (Yamamoto et al., 2001). Much of this work has been driven by gene therapy and agricultural genetic research. The most commonly utilized technique for development of mouse transgenic models is the tetracycline (tet)-inducible promoter. Two versions are commercially available so that an investigator can select a transgene promoter that is always activated until tet is added, or always inactivated until tet is added (Backman et al., 2004). Tetracycline (or the analogue doxycycline) can simply be added to the transgenic mouse's water supply to induce the promoter to express the transgene or turn it off. Another benefit of this technique is its capacity to regulate the activity of the promoter quantitatively, that is, by adding more or less tet to the water. Inducible promoters provide a powerful opportunity to address cause-and-effect relationships between transgene expression and resulting mouse phenotypes, including the roles of development and plasticity. Additional resources describing the theory and methodology of transgenesis, including more detailed protocols, include Jackson and Abbott (2000), Hofker and Deursen
(2002), Nagy et al. (2002), Pinkert (2002), and Houdebine
Compared with homologous recombination-mediated gene knockouts, only a relatively small number of studies have used transgenically expressed ion channels to study epilepsy and neuronal excitability in mice (Table 1). Some examples include the following:
In 1999 Sutherland et al. created three transgenic mouse lines with a human HPRT promoter driving the CNS-specific overexpression of an Aplysia shaker-type K+ channel cDNA with a small 11-amino acid epitope tag. Transgene expression was detected in neurons, but not glia, in several brain areas, including the neocortex, hippocampus, and cerebellum. Electroencephalographic (EEG) activity from adult neocortex showed spontaneous cortical discharges similar to those seen in spike-wave epilepsy. Because overexpression of shaker K+ channel currents in Aplysia neurons shortened action potential duration, enhanced the hyper-polarizing afterpotential, and depressed transmitter release from terminals (Kaang et al., 1992), the presence of an epilepsy phenotype in transgenic mice expressing the same gene was surprising. Further analysis revealed extensively
Phenotype or syndrome*
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