Identification of Ectopic Gene Expression

After delivery of the desired genes into cells, identification of their proper expression is necessary. The general strategies for identification (Chalfie et al. 1994; Kohara et al. 2001; Yu and Rasenick 2002; Yu et al. 2002b) include

1. The measurement of some functional changes elicited by gene expression in targeted cells. If an enzyme is expressed, this would involve measuring the activity of that enzyme.

2. The detection of the proteins coded by the desired genes by techniques relying on antibodies, such as western blot, immunoprecipitation, and immunocytochemistry. Expression of the desired genes tagged with some epitopes, such as HA, GST, and His-tag, can also be determined with antibodies directed against these epitopes.

3. The use of green fluorescent protein (GFP) as an indicator. GFP, a protein from the jellyfish Aequorea victoria, can be either cotransfected with the desired genes or fused with the desired genes before incorporation into cells. The fluorescence from GFP is easy to detect using fluorescent microscopy, and this allows the monitoring of gene expression in living cells. Furthermore, if GFP is fused with the gene of interest, its fluorescence provides a useful tool for studying the function and cellular localization of proteins coded by the genes of interest.

At this point, several different "colors" have been developed through mutation of the initial GFP gene (Shaner et al. 2005). Depending on the wavelengths of excitation, they can be used to localize multiple protein species, or fluorescence resonance energy transfer (FRET) can be used to demonstrate that two target proteins are in close (< 10 nm) proximity. FRET uses two fluors with little spectral overlap and depends on the emission of one of the fluors exciting the other.

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