What factors affect the method that should be chosen to engineer a cell line that upregulates an endogenous protein? I am mostly asking permanent or long-term expression of nuclear proteins in mammalian cell lines, but more selection criteria for more applications would be appreciated too.
As I understand it, the possible methods are the following:
- Target the regulatory factors already present in (or adjacent to) the gene of interest responsible for the protein, possibly targeting known variants that express a phenotype associated with high protein expression
- Introduce new regulatory elements to improve transcription and translation
- If the cell line is immortalized, expand and select for higher expression of the target protein
- Introduce a non-integrative element that expresses the mRNA transcript variant
Are there any factors regarding the protein, gene, or cell type that make one of these better or worse? And are there any methods I'm not considering?