Inducible Knockdown Cells

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

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

Related resources and publications

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Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Front Immunol

Dec 2023

Highlighted article

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CCX559 is a potent orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity

PLoS One

Apr 2023

Highlighted article

read the comment

ERα-36 regulates progesterone receptor activity in breast cancer

Breast Cancer Res

Aug 2020

T cells expressing a chimeric-PD1-Dap10-CD3zeta receptor reduce tumor burden in multiple murine syngeneic models of solid cancer

Immunology

Mar 2020

Tumor Vessel Normalization, Immunostimulatory Reprogramming, and Improved Survival in Glioblastoma with Combined Inhibition of PD-1, Angiopoietin-2, and VEGF

Cancer Immunol Res

Oct 2019

SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Nat Immunol

Aug 2019

iPS technology: Four factors that will change the world… some day

iPS technology

5 min read

Nov 2017

Improving in vitro models: the coming of age of organoids

Organoids

6 min read

Mar 2018

Targeted protein degradation: new promises for “undruggable” diseases (SMASh Part 1)

Smash technology

6 min read

Feb 2021

Degradation tag systems: a good way to SMASh your target (Part 2)

Smash technology

6 min read

Apr 2021

A new promising family of small molecules regulating the PD-L1/PD-1 signaling pathway

Humanized PD-1/PD-L1 mouse and cell line

10 min read

Jul 2022

CCX559 is a potent, orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity

Humanized ICP mouse and cell lines

9 min read

Jun 2023

Development of a new hSIRPα-specific radiotracer for non-invasive PET imaging using genOway’s double humanized genO-hCD47/hSIRPα mouse and genO-MC38-hPD-L1-hCD47-LZ cell line

Targeting the human CD47/SIRPα axis

10 min read

Mar 2024

No results

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