Inducible Knockdown Cell Lines
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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).
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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
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1) MC38-hPDL1 tagged lines express human PD-L1
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. |
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2. Human PD-L1 expression is reversibly shut down upon drug treatment
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:
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For bio-pharmaceutical research & development:
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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.
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