This is important in determining physiological effects A stable transfection allows the plasmid to remain in the cell genome, in contrast to transient transfection where expression is limited due to eventual degradation. The plasmid integrates into the genome of the transfected cell, and becomes part of the cell line, passing on during replication8. Different reagents and other physical tools involving electrochemical changes can be used for transfection, and efficiency depends on the specific cell type. This experiment uses the Lipofectamine transfection agent that forms liposomes which fuse with the plasma membrane by electrochemical changes and allows plasmid passage9. Co-transfection with a marker gene with antibiotic resistance is common. Stability is evident when adding toxins for selective pressure ensures that only the stable ones can proliferate in further cultivations10. In this experiment, we use the Geneticin (G418) toxin agent for isolating drugs with potential antagonism. G418 inhibits polypeptide synthesis (400 μg/mL is recommended). G418 resistance involves the addition of a Tn5 gene that encodes for an …show more content…
Screening for an antagonist needs the use of an active threshold for maximum activity. The active threshold is arbitrary, but 3*standard deviation (3SD) from the control same mean and inhibition is expressed as a percentage. Differences in threshold level choices arise from different experiment targets or desire for accuracy. A lower threshold gives more compounds to select as potential antagonists but increases the probability of false-positives. A higher threshold is stricter and can result in false-negatives. Generally, reduced data variability in assaying provides greater accuracy13. In this experiment, a screen is set for hGPCRx antagonists quantification using an active threshold. Hits should be evaluated for false-positives or