Which marker should I put in my vector?
Choosing an appropriate selection marker is a critical step while designing a vector for ensuring experimental success. A selection marker enables the experimenter to successfully identify cells that have been positively transfected or transduced with the vector.
Two types of markers are commonly used for selection of positively transfected/transduced cells – drug-selection markers and fluorescent protein markers. VectorBuilder offers a variety of drug-selection markers on its vectors, such as puromycin (Puro), neomycin (Neo), hygromycin B (Hygro) and blasticidin (Bsd) which function by conferring resistance against the respective antibiotic in cells carrying the vector with the marker. Cells not carrying the vector on the other hand are non-resistant to the antibiotic and are therefore, killed in its presence. Fluorescent markers on the other hand enable researchers to select for positively transfected/transduced cells by visualization under a fluorescence microscope or by fluorescence activated cell sorting (FACs).
Sensitivity towards a drug-selection marker can vary significantly from one cell type to another. While some cell types may naturally have some degree of resistance to certain antibiotics without the resistance gene, certain cell types are sensitive to certain antibiotics even with the resistance gene. Therefore, different drug-selection markers should be tested to find the optimal one for your cell type. In general, we have found that puromycin kills non-resistant cells faster and more consistently than other antibiotics. For this reason, we recommend puromycin for most cell types.
Once the optimal antibiotic has been selected, it is also important to determine the optimal antibiotic concentration and the duration of selection required to kill the target cells by performing an antibiotic kill curve. This ensures the presence of all positively transfected/transduced cells in the experimental cell population, thereby reducing variability and helps in the establishment of a stable cell line if needed for experiments requiring long-term gene expression. The table below lists the recommended concentration and selection duration of commonly used drugs in common cell lines.
|Antibiotics||Cell line||Recommended concentration||Recommended duration|
|Puromycin||293T||1-2 ug/ml||3-5 days|
|Geneticin (G418)||HT1080||500-1000 ug/ml||7-11 days|
|Blasticidin||293T||5-15 ug/ml||7-11 days|
|Hygromycin B||293T||100-200 ug/ml||5-7 days|
While drug-selection markers are suitable for experiments requiring long-term gene expression through the establishment of a stable cell population which can be time consuming, fluorescent markers on the other hand provide a relatively simple and quicker alternative for selecting positively transfected/transduced cells by visualization under a fluorescence microscope. Selection of an ideal fluorescent marker for your vector depends on several factors such as whether it is single-color or multi-color experiment, brightness of the fluorescent protein, maturation time of the protein and sensitivity of the target cells towards toxicity and protein aggregation observed with certain fluorescent proteins.
While incorporating either a drug-selection or a fluorescent marker in the vector is the most commonly used approach, adding a dual-selection cassette consisting of both a drug-selection marker as well as a fluorescent marker provides a highly versatile and efficient method for selecting positively transfected or transduced cells. However, the use of a dual-selection cassette is sometimes restricted by the limited cargo capacity of the vector being used such as observed with viral vectors. VectorBuilder offers a variety of single as well as dual-selection cassettes to provide you with the flexibility to choose the right selection marker suitable for your experiment.