Academic journal article Genetics

Temporal Control of Cell-Specific Transgene Expression in Caenorhabditis Elegans

Academic journal article Genetics

Temporal Control of Cell-Specific Transgene Expression in Caenorhabditis Elegans

Article excerpt

ABSTRACT

Cell-specific promoters allow only spatial control of transgene expression in Caenorhabditis elegans. We describe a method, using cell-specific rescue of heat-shock factor-1 (hsf-1) mutants, that allows spatial and temporal regulation of transgene expression. We demonstrate the utility of this method for timed reporter gene expression and for temporal studies of gene function.

THE ability to manipulate transgene expression in animals has facilitated the study of many biological processes. 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. For example, restriction of transgene expression to a specific cell type can allow determination of whether gene function is cell autonomous or nonautonomous. Likewise, temporal control of transgene expression is critical for determining whether the function of a gene of interest is required continuously or only during specific times.

Several strategies that allow control of both where and when transgenes are expressed have been developed. Spatial resolution is often achieved by the use of tissueor cell-specific promoters, and timing of transgene expression can be controlled by introduction, or withdrawal, of small-molecule inducers, or inhibitors, of gene expression. For example, a fusion protein consisting of the tetracycline repressor fused to the transcription activation domain of VP16 (TetR-VP16), and expressed using a tissue-specific promoter, can promote tissue-specific expression of genes bearing tetO operator sequences. Addition of the cell-permeable ligand tetracycline, which binds to and prevents TetR-VP16 from binding tetO (Gossen and Bujard 1992; Gossen et al. 1995), can be used to extinguish expression at specific times. Similarly, Cre recombinase fused to the estrogen receptor, and expressed in specific tissues, can be temporally activated by the addition of tamoxifen (Feil et al. 1996).

An alternative to using small molecules for temporal control is afforded by proteins whose functions are regulated by temperature. For example, in Drosophila, the GAL4/UAS system has been used to control celltype- specific transgene expression(Brand and Perrimon 1993). McGuire et al. (2003) modified this system to achieve temporal control of GAL4-driven expression by introducing a temperature-sensitive form of GAL80, a GAL4 inhibitor, into transgenic flies. At permissive temperatures, animals fail to express the transgene because GAL80 inhibits GAL4. Shifting to nonpermissive temperatures relieves GAL80 inhibition and allows transgene expression to proceed.

Another strategy for temporal control of transgene expression exploits the heat-shock response, a temperaturedependent stress defense mechanism. The heat-shock response is mediated by heat-shock factor (HSF), a transcription factor that is synthesized constitutively, but remains latent during unstressed conditions (Lis and Wu 1993). In response to heat stress, HSF trimerizes and binds with high affinity to promoters containing specific binding elements, leading to the transcription of heatshock proteins (Pelham 1982; Westwood et al. 1991). Thus, transgenes containing HSF-binding elements can be induced, albeit with little cellular specificity, following a temperature shift. Attempts to lend spatial resolution to the heat-shock response by using heated needles (Monsma et al. 1988; Vekris et al. 2000) or focused laser microbeams (Stringham and Candido 1993; Halfon et al. 1997) to trigger the response in specific cells have been described. However, these physical methods for spatially restricted heat-shock delivery are labor intensive and potentially damaging to cells, explaining, in part, why they are not in common use.

Currently, no facile approach is available for combined spatial and temporal regulation of transgene expression in Caenorhabditis elegans. …

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