1
$\begingroup$

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?

$\endgroup$

1 Answer 1

1
$\begingroup$

This is one of those questions that needs to be determined empirically, as it is highly dependent on the protein in question. For any one system, the best method to use needs to be determined by trial and observation. Upregulation and over-expression can be problematic, depending on the system you are targeting. Check the literature to see what others have used.

You are missing a few methods, namely those that use a lower expression cf. the already determined level in your unaltered line:

In many cases it is possible to find a cell-line that has a naturally higher or lower expression of the protein that can be used experimentally to compare to another that has a lower/higher expression, or use/generate a Knock-Out (KO) version of the cell line. Generation of KO is generally easier than introduction of a gene for stable expression if you are aiming for integration into the genome. The exception here is where the gene is used on a mobile element like a plasmid and is continually selected for using selective marker for over-expression. Investigate CRISPR/Cas systems for these, as well as viral vector methods (lentivirus, adenovirus etc.)

You can also use methods such as siRNA to target mRNA for degradation, but this is not stable, needs to be done individually for each experiment, and may or may not work for your system.

$\endgroup$

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .