A reporter gene is used to follow gene expression in vivo. It is a fusion between all or part of a gene of interest with another gene whose product is easy to detect or measure qualitatively and/or quantitatively. Most often, the researcher will choose to use a reporter gene if the product of the gene of interest is difficult to assay or detect. Thus, the reporter gene product acts as a surrogate.
Reporter gene (ORF only)
Promoter fusion YGll promoter
In-frame fusion YGll promoter
A fusion gene between the gene of interest and the reporter gene can include only the upstream promoter of the gene of interest or part or all of its ORF. If a coding region is included, then the sequence at the fusion junction must maintain the correct reading frame so that a single ORF is produced that encodes a fusion of the two proteins. This is shown in Figure 1.6. Fusion gene constructions are often carried on plasmid vectors but they can also be integrated into chromosomes, depending on the needs of the experiment.
There are several commonly used reporter genes including lacZ (encoding 6-galactosidase from E. coli), CAT (encoding chloramphenicol acetyltransferase, the bacterial protein providing chloramphenicol resistance), luciferase gene (encoding the phosphorescent protein from firefly), and GFP [encoding green fluorescent protein (GFP) from a jellyfish]. To be useful, the host organism must not encode a protein with the same activity, otherwise one could not be sure whether one was observing the activity of the endogenous protein or the reporter protein. Using a variety of techniques, these proteins can be measured either in vivo or in vitro. ¡3-Galactosidase can be assayed in vitro using cell extracts by measuring the rate of hydrolysis of an uncolored compound called ONPG to a yellow dye or another uncolored compound called X-gal to a blue dye. The X-gal reaction is particularly useful because is can be done on whole cells in tissues or colonies growing in a petri dish. Cells expressing the /3-galactosidase reporter will be bright blue. GFP is very useful for determining the subcellular location of a protein and the type of fusion used for this analysis is an in-frame fusion between the full-length gene of interest and the GFP gene (discussed in detail in Chapter 2). Alternately, portions of the gene of interest can be fused to GFP to localize the portion of the protein of interest responsible for targeting the protein to a particular subcellular compartment. There are many other uses of these types of construction.
A variety of E. coli/yeast shuttle vectors are available for the construction of fusion genes. These contain a multiple cloning sequence at the junction site of the fusion. The DNA sequence to be fused to the vector gene, whether it is a promoter or an ORF, is typically amplified by PCR using primers that place appropriate restriction sites at the ends of the fragment. The fragment is then cloned into the multiple cloning site to create the fusion. PCR-based methods are also available for creating fusions at sites in the genome and these are described in Chapter 2 (Longtine et al., 1998).
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