The present invention relates generally to chemical-inducible system and to methods of use in transgenic animals. More specifically, the present invention relates to a chimeric transcription factor that binds to a ligand and functions in ligand-dependent manner to induce expression of genes of interest under the control of a synthetic operator-promoter sequence. The expression of genes of interest can be tightly controlled by adding or removing the ligand.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Transgenic organisms that express or misexpress various transgenes are commonly generated for biological research and biotechnology. However it is often problematic to generate or study the transgenic organisms that carry and express certain transgenes. For example, i) expression of a transgene which causes lethality or infertility makes it is impossible to create transgenic animals carrying a given transgene; ii) the developmental or other effects caused by expression of a given transgene in the earlier development makes it is impossible or problematic to unambiguously study the effects that occur later in development; and iii) continuous expression of the transgene after a certain time point may be undesirable.
Also, many developmentally regulated genes are re-deployed at different times of development in different cell types. Therefore, in order to dissect their unique functions in each context in a precise manner, methods for manipulation and spatiotemporal control of gene expression are essential. Using a simple promoter to drive target gene expression is the most commonly used strategy in transgenic fish and other vertebrate species. However, the number of well-characterized tissue-specific promoters is very limited. Furthermore, constitutive and ubiquitous expression is often not adequate or disadvantageous, for example, when gene expression at earlier stages of development causes severe effects, which obscure the roles of this gene at later stages or when expression of the transgene cause infertility or premature lethality, hindering generation and maintenance of transgenic animals.
One way to address these problems is by generating two independent transgenic lines, the “effector (target)” line, carrying a transcriptionally silent gene of interest, and the “driver” line, expressing a transcription activator which can induce expression of the gene of interest in double transgenic hybrid driver/effector animals (e.g. Gal4/UAS system (Davison et al., 2007; Scott et al., 2007)). The use of gene- and enhancer-trap screening can allow the generation of a large number of driver lines with various expression patterns of a transcription activator (Kotani et al., 2006; Parinov et al., 2004; Scott et al., 2007). However, it is not feasible to cover all required spatiotemporal patterns even in large-scale enhancer-trap screens. Therefore, improved techniques that permit switching gene expression on and off when required, would allow more experimental flexibility and better control of expression.
Heat shock promoters have been utilized in fish to induce ubiquitous gene expression at specific time points by exposure to heat (Bajoghli et al., 2004). For example, heat-inducible expression of the dominant-negative form of the FGF receptor driven by a zebrafish hsp70 was used to study the roles of Fgf signaling during regeneration (Lee et al., 2005; Lepilina et al., 2006). However, heat shock promoters allow relatively low inducibility and lack spatial control of transgene expression. Also, using lasers to induce cell-specific gene expression (Halloran et al., 2000) is technically difficult and applicable only on a small scale.
Non-inducible gene expression systems utilizing the LexA DNA binding domain have been previously used in invertebrates and in vertebrate cell cultures (Hoshino et al., 2004; Lai and Lee, 2006; Nettelbeck et al., 1998; Szuts and Bienz, 2000).
Chemical-inducible techniques allow temporal control of gene expression combined with the spatial control of tissue-specific promoters. Furthermore, binary inducible expression systems allow a combination of inducibility with the power of enhancer trap screening (Nicholson et al., 2008). Chemical inducible expression systems have been previously applied to study gene functions at various stages of development. For example, “tet-on” inducible system was used to dissect multiple roles of genes during chicken somitogenesis, which would not be possible using a conventional promoter because the phenotypes caused by the early expression of some genes precluded studying the effects at the later stages (Watanabe et al., 2007). Mifepristone-inducible expression of FGF-3 during postnatal development, and in the adult tissues was used to assess the complex temporal roles of the FGF signaling in organ development, adult physiology and tumor development in transgenic mice, because manipulation of FGF signaling at earlier stages caused abnormal development and neonatal lethality (Ngan et al., 2002; Zhao et al., 2001). In another example, inducible expression of dominant negative thyroid hormone receptor was used to determine the developmental periods within which thyroid hormone controls specific aspects of Xenopus morphogenesis (Das and Brown, 2004). Inducible expression used in this example also helped to circumvent the severe developmental abnormalities and death caused by expression of the dominant negative receptor using conventional promoters.
Hormone-responsive transcriptional activators have been used previously in ligand-inducible strategies to regulate target gene expression. For example, a chimeric transactivator consisting of a mutated progesterone receptor ligand-binding domain fused to the HSV VP16 transactivation domain and the yeast GAL4 DNA-binding domain (DBD) has been shown to transactivate UAS-controlled target genes only in the presence of mifepristone/RU-486 (Wang et al., 1994). A similar chimeric transactivator GLp65, which contains the activation domain of the human p65 protein (instead of VP16), has been used for inducible expression of target genes in mammalian cells (Burcin et al., 1999). Mifepristone-inducible GAL4/UAS-based techniques have been successfully used in transgenic mice (Kellendonk et al., 1999; Ngan et al., 2002; Pierson et al., 2000; Wang et al., 1997a; Zhao et al., 2001), in Xenopus (Das and Brown, 2004) and in Drosophila (Nicholson et al., 2008). Although the Gal4/UAS system has been widely utilized previously, some reports suggest that high level of GAL4 expression can be toxic and causes developmental defects (Habets et al., 2003; Kramer and Staveley, 2003; Scott et al., 2007).
One alternative to GAL4/UAS-based systems is to use the DNA-binding domain from the bacterial repressor LexA coupled with the specific operator DNA fragment/s. Since the structure of LexA DBD does not resemble those of eukaryotic transcription factors (Oertel-Buchheit et al., 1992), it is less likely to bind to the cis-elements of endogenous promoters. It was previously utilized in estradiol-inducible gene expression systems, developed for use in transgenic plants, since plants lack endogenous estrogen hormones (Guo et al., 2003; Zuo et al., 2000). This expression system caused no apparent toxicity in transgenic plants.
Although several chemical-inducible systems have been developed for use in mammals and mammalian cells, there has been only one recent publication in zebrafish. In this report, a tebufenozide-inducible system was tested in transient assay (Esengil et al., 2007). It utilized a chimeric transcription factor containing GAL4 DNA-binding and dimerization domains, VP16 activation domain and ecdysone receptor (EcR) ligand-binding domain. However, this chemical-inducible system has not been used yet for generation of true transgenic animals or for generating driver/effector lines, so the efficacy of this system is not clear.
Thus, there is still a need for new genetic systems that would make it possible to effectively control transgene expression by switching it on or off when required and to drive the expression only in the desired tissues or cells.