Cloning & Expression
Cloning and gene expression technologies play a pivotal role in research across various scientific disciplines, enabling the investigation of a broad spectrum of biological questions. These technologies help in understanding gene function, analyzing molecular pathways, studying embryonic development, exploring disease mechanisms, and facilitating the bioprocessing of biologics and therapeutics. Once a gene or genetic sequence is identified, researchers must carefully select the most appropriate molecular cloning strategy and cell-based protein expression system tailored to the specific needs of their application. For more insights on gene expression using CRISPR technology or gene silencing with RNAi reagents, please visit our dedicated gene expression and gene silencing page.
Overview
Protein Expression Vectors
Plasmids, or protein expression vectors, are circular pieces of DNA that contain numerous functional elements for cloning and expressing the protein of interest. The selection of the appropriate expression vector is partially determined by the type of protein, the organism that will express the protein, and the specific application and scientific questions the researcher aims to address. The wide variety of expression vectors available allows researchers to choose which cloning, clone selection, and protein expression elements are best for their study. Once the gene sequence of interest and the expression vector is selected, researchers commonly use nucleases to precisely cut the vector at specific sites to allow the genetic sequence of interest to be cloned in-frame and ultimately expressed in a host organism. Importantly, researchers will commonly use chemically competent cells and bacterial transformation to host the recombinant DNA plasmids as they are stable when stored frozen at -70 °C.
How to Transfect Cells
Cell transfection refers to the process of introducing DNA or RNA, or proteins into eukaryotic cells. A variety of physical, chemical, and biological technologies are available to researchers to accomplish this part of the protein expression workflow. Physical methods, such as electroporation, or chemical methods, including calcium phosphate (CaPi), polyethylenimine (PEI), and lipofection reagents are all commonly used by researchers. Viral transduction systems, including lentiviral, oncoretroviral, and adenoviral are also available to scientists. However, the use of viral delivery methods often requires additional containment and monitoring for biosafety reasons.
Protein Expression Systems
Various organisms are used by researchers to express their protein of interest. Bacteria are commonly used by researchers as they efficiently uptake expression vectors, have fast doubling times, and can produce high quantities of recombinant protein under optimal conditions. However, as bacteria are prokaryotic organisms, they are not able to provide protein post-translational modifications that some proteins require. Eukaryotic cells, including yeast and mammalian cell lines, facilitate protein post-translational modification(s) and are also commonly used by researchers. The choice of the best cell line for recombinant protein expression and the protein expression strategy is highly dependent on the specific application needs and questions the scientist aims to address.
Document Search
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