Good gene editing controls are a critical tool for troubleshooting and ensuring that the transfer worked according to plan, especially due to the high sensitivity of gene transfer experiments. RNA interference (RNAi) and reporter gene experiments assess the activity and effects of certain genes. These techniques require controls to test transfection efficiency, knockdown efficiency (RNAi), and whether conditions are still optimal (reporter genes).
Gene Transfer Controls
Gene transfer introduces specific genetic sequences into a new host cell. This process tests whether overexpression of genetic material alters cellular functions, or if the expression of a particular gene is possible in a certain cellular system.
When choosing your gene transfer control, there are a few factors to consider. For instance, there is a difference in procedure between bacterial and mammalian cells.
When transforming bacteria, it is common to perform an additional transformation with a control vector. Vectors such as the pUC18 plasmid or the pUC19 vector are normally used for such cases. These cloning vectors, given their multiple cloning and restriction sites, can easily introduce foreign DNA into the plasmid. Antibiotics can be used as a control for transformed bacteria. This is done with a small plasmid which carries an appropriate antibiotic resistance. In addition to this, the X-gal system provides an extra layer of control by color-coding successfully transformed colonies that carry the right insert. One key example of this process is the blue-white screening technique, as seen with the pUC19 plasmid system.
Antiobiotics can also be used as controls for transformed mammalian cells. However, cells growing on antibiotics might display physiological differences that could influence your experiments. An alternative gene transfer control involves vectors which express a fluorescent protein alongside with the gene of interest. This can also be used for co-transfection of two vectors, using multiple fluorescent proteins, or for automatic selection of successful transfection.
Reporter Gene / siRNA Controls
Reporter control siRNAs ensure high reporter gene knockdown of genes such as GFP and luciferase, which indicate gene expression. Knockdown of these genes allows you to visualise when conditions are suboptimal and need to be changed; this is indicated by test samples fading to a similar level of the control.
RNAi controls are able to fully inhibit your RNA of choice or leave it completely unaffected, and therefore give a measure of the efficacy of your technique of inhibition.
Compare Gene Editing Controls
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