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Inducible Knockdown cells
Cell line generation

Inducible Knockdown cells

Through a single genetic modification, the addition of a self-cleaving degron tag to a protein of interest allows its inducible, and reversible, degradation upon treatment.

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An inducible Knockdown cell line defines a model in which a specific target of interest can be shut down upon treatment.

We use SMASh technology to generate drug inducible protein degradation models. Through a single genetic modification, the addition of a self-cleaving degron tag to a protein of interest allows its inducible, and reversible, degradation upon treatment (further reading).

genOway has an exclusive license for the use of the patented SMASh technology. (See press release)

Strengths of inducible Knockdown cell lines

  • Inducible and reversible system
  • One cell line = several models with different level of expression of the protein of interest
  • Reliable: each cell line is matched with an isogenic control cell clone
  • Feasible in all genetic backgrounds and cell types

Limitations of inducible Knockdown cell lines

  • Efficiency of down regulation depends on the target and cell line of interest
  • Genetic modification of the gene of interest may deregulate neighboring genes, inducing a non-specific phenotype
  • Challenging in cells with high polyploidy

→  Risks can be strongly minimized by applying in-depth bio-informatic, genetic, and bibliographic analyses
→ An alternative model is a functional Knockout by introducing a point mutation to produce an inactive protein

Proof of concept

Immune checkpoint PD-L1 has been identified as a therapeutic target in immuno-oncology and different treatments have been successfully developed to target this protein in oncology.

We developed mouse tumor MC38 cells invalidated for mouse Pd-l1 and overexpressing a SMASh-tagged form of human PD-L1 to reversibly shut down its expression in these cells, thus obtaining cells entirely devoid of PD-L1 upon drug treatment.

Two cell lines developed:

  • MC38-SMASh-hPD-L1, with the tag inserted in N-term of the targeted protein
  • MC38-hPD-L1-SMASh, with the tag inserted in C-term of the targeted protein

Applications

For academic research:

  • Determine and study main functions of the gene and/or protein
  • Loss-of-gene-function studies‍
  • In vitro recapitulation of a human disease

For bio-pharmaceutical research & development:

  • In vitro target validation
  • Mimic human disease for drug studies and screenings
  • Positive control for antagonist drug screening‍
  • In vitro model for targeted protein degradation

Validation

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