Several steps in the gene expression process may be modulated, including the transcription step and translation step and the post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in the organism.
A more modern low-throughput approach for measuring mRNA abundance is real-time polymerase chain reaction (The term RT-PCR is used to refer to both reverse transcription PCR as well as real-time PCR, which is also known as quantitative RT-PCR or quantitative PCR (qPCR). With a carefully constructed standard curve qPCR can produce an absolute measurement such as number of copies of mRNA per nanolitre of homogenized tissue. The lower level of noise in data obtained via qPCR often makes this the method of choice, but the price of the required equipment and reagents can be prohibitive.
In addition to low-throughput methods, transcript levels for many genes at once (expression profiling) can be measured with DNA microarray technology or "tag based" technologies like Serial analysis of gene expression (SAGE) or the more advanced version SuperSAGE, which can provide a relative measure of the cellular concentration of different messenger RNAs. Recent advances in microarray technology allow for the quantification, on a single array, of transcript levels for every known gene in the human genome. The great advantage of tag-based methods is the "open architecture", allowing for the exact measurement of any transcript, known or unknown. Especially SuperSAGE recommends itself therefore also for studying organisms with unknown genomes.
Protein levels themselves can be estimated by a number of means. The most commonly used method is to perform a Western blot against the protein of interest, whereby cellular lysate is separated on a polyacrylamide gel and then probed with an antibody to the protein of interest. The antibody can either be conjugated to a fluorophore or to horseradish peroxidase for imaging or quantification. Another commonly used method for assaying the amount of a particular protein in a cell is to fuse a copy of the protein to a reporter gene such as Green fluorescent protein, which can be directly imaged using a fluorescent microscope. Because it is very difficult to clone a GFP-fused protein into its native location in the genome, however, this method often cannot be used to measure endogenous regulatory mechanisms (GFP-fusions are therefore most often expressed on extra-genomic DNA such as an expression vector). Fusing a target protein to a reporter can also change the protein's behavior, including its cellular localization and expression level.
The pattern of detection of a gene or gene product may be described using terms such as facultative, constitutive, circadian, cyclic, housekeeping, or inducible.
In addition to these biological tools, certain naturally observed configurations of DNA (genes, promoters, enhancers, repressors) and the associated machinery itself are referred to as an expression system, as in the simple repressor 'switch' expression system in Lambda phage. It is these natural expression systems that inspire artificial expression systems, (such as the Tet-on and Tet-off expression systems).
Each expression system has distinct advantages and liabilities, and may be named after the host, the DNA source or the delivery mechanism for the genetic material. For example, common expression systems include bacteria (such as E.coli), yeast (such as S.cerevisiae), plasmid, artificial chromosomes, phage (such as lambda), cell lines, or virus (such as baculovirus, retrovirus, adenovirus).
Often, the DNA sequence for a protein of interest will be cloned or subcloned into a plasmid containing the lac promoter, which is then transformed into the bacterium Escherichia coli. Addition of IPTG (a lactose analog) causes the bacteria to express the protein of interest. However, this strategy does not always yield functional protein, in which case, other organisms or tissue cultures may be more effective. For example, the yeast Saccharomyces cerevisiae is often preferred to bacteria for proteins that undergo extensive posttranslational modification. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination.