Question

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?

Answer 1

Choosing a method to upregulate an endogenous protein in a cell line depends on several biological factors including gene and protein function, regulatory mechanisms, and cell type. A detailed understanding of gene regulation and biotechnological tools like CRISPR-Cas and epigenetic factors facilitate informed method selection. Consideration of potential long-term expression outcomes, such as oncogenesis, is crucial in the decision-making process.

Step-by-step explanation:

Factors affecting the choice of method to engineer a cell line for upregulating an endogenous protein include the protein's function, gene structure, cell type, and desired duration of expression. Targeting regulatory factors involves manipulating the protein's own gene regulation, while introducing new regulatory elements can enhance transcription and translation efficiency. It's important to recognize that modifications like the insertion of non-integrative elements might provide transient expression, whereas targeting endogenous gene regulation could achieve a more stable outcome. Consideration should be given to post-transcriptional and post-translational modifications and the potential for unintended effects like oncogenesis when choosing a method.

Biotechnology tools, like CRISPR-Cas, enable precise alterations to gene expression, while epigenetic factors like histone modification also play a critical role in regulating gene accessibility. Ultimately, the choice of engineering strategy depends on a detailed understanding of the gene's regulation, the specificities of the protein involved, and the context within which the cell line will be used. Other methods not covered in the question may involve RNA interference or the use of specific drugs to modulate protein expression levels.