A useful complement to knocking out and reducing gene expression is to overexpress or misexpress the gene of interest. This approach can answer questions such as whether a gene is limiting for a certain process or whether precise temporal and spatial regulation of its expression is required for normal function.
Constitutive/Tissue-specific Promoter Fusions
The simplest way to overexpress and misexpress a gene is often to fuse the gene of interest to a so-called constitutive promote^ such as the CaMV 35S promoter (Odell et al. 1985). Several vectors contain a CaMV 35S/terminator cassette into which a gene of interest can be cloned, e.g., pART7/pART27 (Gleave 1992), pCGN18 (Jack et al. 1994), and pCHF3 (Neff et al. 1999).
Fusions to the CaMV 35S or a similar promoter can, however, have several limitations. First, the CaMV 35S promoter is not truly constitutive in the sense that expression in all tissues is the same. Thus, it should be determined whether or not expression in the tissue of interest is indeed high. Second, constitutive expression may be detrimental to the plant, and it may prove impossible to obtain stable transformants with high levels of widespread overexpression. In either case, the use of tissue-specific promoters may be advisable. Alternatively, a two-component or inducible system, as described in the following section, can be used.
Two-component Systems for Tissue-specific Misexpression
If expression from a constitutive promoter, or even from a tissue-specific promoter, proves to be lethal or to cause sterility, an alternative sys tem can be used, in which two otherwise inactive components are combined to activate the gene of interest. Several such systems have been developed for Arabidopsis, using chimeras of bacterial, viral, yeast, or mammalian transcription factors. The first component is an activator in
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