Inducible Knockdown Cell Lines

 

Get supplemental information, a quote, and estimated timeframe to generate your inducible Knockdown cell line.

 

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)

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

1) MC38-hPDL1 tagged lines express human PD-L1

MC38-SMASh-hPD-L1-Img01

Human PD-L1 expression was analyzed by flow cytometry. MC38 parental line was used as a negative control. Human colon carcinoma cell line RKO was used as a positive control. Both tagged cell lines express detectable levels of human PD-L1. Note that the position of the SMASh-tag can impact the targeted protein’s level of expression, as MC38-hPD-L1-SMASh cells express higher levels of human PD-L1 than MC38-SMASh-hPD-L1.

2. Human PD-L1 expression is reversibly shut down upon drug treatment

MC38-SMASh-hPD-L1-Img02

Drug-inducible regulation of human PD-L1 expression. MC38-SMASh-PD-L1 and MC38-hPD-L1-SMASh cells were treated with asunaprevir (ASV) for 8 days, then ASV was removed for the following 7 days. Human PD-L1 expression was assessed by flow cytometry. At Day 4, ASV treatment efficiently shut down expression of human PD-L1 (A). Human PD-L1 shut down was efficiently reversed at Day 15 after removal of the drug (B). Although both tagged lines responded to ASV, human PD-L1 expression levels were lower upon treatment in MC38-hPD-L1-SMASh cells than in MC38-SMASh-hPD-L1 cells (C). Reversibility was observed as early as 1 day after drug withdrawal. MC38 stably expressing a non-tagged human PD-L1 did not respond to drug treatment (data not shown).

 

Typical applications for inducible Knockdown cell lines


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

Strengths and limitations of inducible Knockdown cell lines

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  • 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
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  • 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

 

References

Hokyung K Chung, Conor L Jacobs, Yunwen Huo, Jin Yang, Stefanie A Krumm, Richard K Plemper, Roger Y Tsien, Michael Z Lin.
Tunable and reversible drug control of protein production via a self-excising degron.
Nat Chem Biol. 2015 Sep.

 

Follow this link if you seek another customized cell line model.