Genes
Genes refer to the genetic information stored by DNA. They have often been regarded as single nodes in a complex network of inherited information. Genes are expressed through cellular machinery, leading to the production of functional products such as proteins. Genes determine all the characteristics of an organism, and are extremely valuable to biological studies and medicinal fields.
Methods of regulating and changing gene expression 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, which contribute to information processing inside the cell whereby overall cellular behavior is determined. Transcription factors are proteins that help activate, or sometimes inhibit, transcription of DNA by binding to particular DNA sequences. They play a major role in gene regulation and cellular differentiation. There are also steps in the general gene expression process which may be modulated, including both transcription and translation (https://www.nature.com/scitable/topicpage/regulation-of-transcription-and-gene-expression-in-1086/).
Expression Vectors
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 transfected with plasmids, viruses (such as a retrovirus), artificial chromosomes, and phages. Other systems include cell lines that have been modified to express desired genes.
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. 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.
Detection
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 a 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 quantify protein expression levels, a Western blot is performed for the detection of 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 quantification is accomplished either via fluorescence or a colorimetric enzymatic tag such as Horse Radish Peroxidase (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.
To directly detect and quantify mRNA levels, a Northern blot can be used. This technique allows scientists to observe its progress at multiple points, like how well it has transferred to the membrane or how intact the RNA sample is. Electrophoresis is first used to separate the RNA strands by size under denaturing conditions. The RNA gets transferred to a membrane and joined with a labeled probe. The advantages to using Northern analysis are being able to easily determine transcript size, identify alternatively spliced transcripts, and compare the abundances of a specific code among all the samples tested. However, the blot does require high quality RNA samples, as even a small amount of degradation can affect the analysis.