For experiments in eukaryotic cells, two commonly used reporter proteins are the enzyme β-galactosidase (β-gal) (see Figure 7–28) and green fluorescent protein (GFP) (see Figure 9–22).
the contribution of individual cis-regulatory sequences in establishing this pattern (see also Figure 7–29).
Reporter genes also allow any protein to be tracked over time in living cells. Here, the reporter gene typically encodes a fluorescent protein, often green fluorescent protein (GFP), the molecule that gives luminescent jellyfish their greenish glow. The GFP is simply attached—in the coding frame—to the protein-coding gene of interest. The resulting GFP fusion protein often behaves in the same way the normal protein does and its location can be monitored by fluorescence microscopy, a topic that is discussed in the next chapter (see Figure 9–25). GFP fusion has become a standard strategy for tracking not only the location but also the movement of specific proteins in living cells. In addition, the use of multiple GFP variants that fluoresce at different wavelengths can provide insights into how different cells interact in a living tissue (Figure 8–61).
In situ Hybridization Can Reveal the Location of mRNAs and Noncoding RNAs
It is also possible to directly observe the time and place that an RNA product of a gene is expressed using in situ hybridization. For protein-coding genes, this strategy often provides the same general information as the reporter gene approaches described above; however, it is crucial for genes whose final product is RNA rather than protein. We encountered in situ hybridization earlier in the chapter (see Figure 8–34); it relies on the basic principles of nucleic acid hybridization. Typically, tissues are gently fixed so that their RNA is retained in an exposed form that can hybridize with a labeled complementary DNA or RNA probe. In this way, the patterns of differential gene expression can be observed in tissues, and the location of specific RNAs can be determined (Figure 8–62). An advantage of in situ hybridization over other approaches is that genetic engineering is not required. Thus, it is often simpler and faster and can be used for genetically intractable species.
Expression of Individual Genes Can Be Measured Using Quantitative RT-PCR
Although reporter genes and in situ hybridization accurately reveal patterns of gene expression, they are not the most powerful methods for quantifying amounts of individual RNAs in cells. We have seen that RNA sequencing can provide information about the relative abundance of different RNA molecules (see Figure 7–3). Here, the number of “sequence reads” (short bits of nucleotide sequence) is proportional to the abundance of the RNA species. But this method is limited to RNAs
Figure 8–61 GFPs that fluoresce at different wavelengths help reveal the connections that individual neurons make within the brain. This image shows differently colored neurons in one region of a mouse brain. The neurons randomly express different combinations of differently colored GFPs (see Figure 9–13), making it possible to distinguish and trace many individual neurons within a population. These images were obtained by genetically engineering the genes for four different fluorescent proteins, each flanked by loxP sites of recombination (see Figure 5–66), and integrating them into the mouse germ line. When crossed to a mouse that produced the Cre recombinase in neuronal cells, the fluorescent protein genes were randomly excised, producing neurons that express many different combinations of the four fluorescent proteins. Over 100 combinations of fluorescent protein can be produced, allowing scientists to distinguish one neuron from the next. The stunning appearance of these labeled neurons has earned these animals the colorful nickname “brainbow mice.” (From J. Livet et al., Nature 450:56–62, 2007. With permission from Macmillan Publishers Ltd.)