Why isn't my drug-selection marker making my cells drug-resistant?
Drug-selection genes typically require only low to moderate expression to be effective, but in some cases they may be insufficient to confer full drug resistance to cells. Below are some of the most frequent causes for the lack of drug resistance:
The dosage of the drug is too high for your cell line
The response to different drugs varies from cell line to cell line. Before applying a new drug on your cell line, we recommend that you always generate a kill curve (also called “dose response curve”) and use the lowest drug concentration that effectively kills the non-transfected or non-transduced cells. Using an unnecessarily high drug dosage can result in the death of cells expressing drug resistance gene, and can also compromise the health of even resistant cells. Some cells are extremely sensitive to certain drugs, in which case you will have to consider using another drug-selection marker.
The drug-selection marker is driven by a weak promoter
Some promoters are inherently weak (e.g. UBC), and can result in low expression of the drug-selection marker. You can consider using a stronger promoter (e.g. EF1A) to drive expression of the drug-selection marker.
The drug-selection marker is expressed through lentivirus or MMLV
For retroviral vector systems (e.g. lentivirus or MMLV), internal polyadenylation signals cannot be present between the LTRs, as this would inhibit virus packaging. Instead, a single polyadenylation signal is present in the 3’LTR. As a result, transcription from the upstream promoter often continues past the end of the upstream ORF, through the downstream promoters and ORFs. This often leads to partial inhibition of expression of the downstream ORF(s). When the downstream ORF is a drug-selection marker, then its expression can be much reduced. You can improve your marker expression as follows:
Consider using a different vector system, such as regular plasmid, adenovirus or adeno-associated virus.
Consider expressing the drug-selection marker in an upstream expression cassette as part of a polycistron. You can use 2A linker to connect the drug-selection marker to your gene of interest. However, this may affect the biological function of the other ORF(s) in the polycistron, may still result in weak drug resistance, and will increase the cost and time of vector construction.
The drug-selection marker is downstream of an IRES
In a polycistronic transcript containing one or more IRES elements, the gene downstream of the IRES is expressed at much lower levels (typically 10-20%) as compared to the first gene in the polycistron. If a drug-selection marker is expressed downstream of an IRES, this reduction in expression may well lead to poor drug resistance. If you have to express a drug-selection marker in a downstream position of a polycistron, you can consider using a 2A linker (e.g. P2A or T2A) instead of IRES. By using 2A, the downstream ORF of a polycistronic transcript is expressed at a level that is comparable to (or only moderately lower than) the upstream ORF. However, 2A also has its pitfalls that could comprise function of the gene of interest. Most notably, the cleavage of the 2A linker leaves behind an extra short peptide on the C terminus of the upstream protein and an extra proline on the N terminus of the downstream protein. This is not an issue in most applications, but under some circumstance, it can comprise protein function.