Genes, Expression Vectors and Detection
Genes have often been regarded as a single node in a complex network, with the expression of the gene being a procedure during which the inherited genetic information of the gene is manufactured into a functioning gene product. In this description, the inheritable information in a gene would be DNA, the RNA molecules would be an intermediate and the actionable gene product is a protein.
Expression of genes and proteins are important tools in research and industrial settings, particularly in the pharmaceutical industry. Not only are genes important for the discovery of therapeutics and preventative treatments like vaccines, but they enable the study of gene regulation, intracellular interactions, antibody generation and protein structure. Proteins produced intracellularly for research and industrial use include antibodies, viral subunits and gene therapy vectors.
A single gene performs a specific function, with the output being the level of protein expression. Some proteins are known as transcription factors, with the function being a type of information processing inside the cell whereby cellular behavior is determined. There are steps in the gene expression process which may be modulated, including both transcription and translation.
A gene expression system is defined as having the molecular mechanism required to transcribe DNA into mRNA and then translate the mRNA into a protein. This definition includes every living cell able to produce protein from a DNA template. A gene expression system is merely a tool in a research laboratory, which may be used for assembling the products of specific genes of interest. Gene expression vectors provide an environment to allow cloned DNA and the host cells to express proteins at a high level.
The natural configurations of DNA together with these biological tools inspire artificial gene expression systems and these systems have both liabilities and advantages. For example, common gene expression systems include bacteria such as plasmids, E.coli, viruses (such as retrovirus), artificial chromosomes, phage and cell lines.
The expression of genes is often regulated after transcription. However, an overall increase in the number of specific mRNA molecules does not necessarily mean that gene expression of the protein will be increased. On the contrary, mRNA concentration has shown to be a poor indicator of protein abundance. The ability to regulate gene expression allows the cell to have control over both structure and function. This is the basis for the environment adaptability cells. It also serves as an argument for morphogenesis and evolutionary changes, as controlling the location and timing of gene expression affects the viability of the cell.
One of the modern approaches to measure mRNA abundance is qRT-PCR. This method uses a standard curve to produce an absolute, real-time measurement of the number of starting copies of mRNA, DNA or miRNA from an extremely small starting concentration of material. This makes it a method of choice especially since optimized reagents are commercially available. This well-established technique allows for the measurement of gene expression in real time detection, permitting both quantitative and qualitative measure of gene silencing effects.
In order to quantitate protein expression levels, the Western blot is a well utilized assay that detects a single protein. Western blots require obtaining an antibody that is highly specific to the target protein and involves three steps. Proteins are first separated by size, and then transferred to a nitrocellulose membrane or solid support, followed by incubation with the antibody specific to the target protein. Imaging and quantitation is accomplished either via fluorescence or a colorimetric enzymatic tag such as HRP. An alternative to traditional membrane transfer is the WES detection system. This automated system enables the researcher to use extremely small sample starting volumes and produce highly sensitive and quantitative protein expression data.
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