How Inducible Systems Work
The Two Component System
Inducible conditional knockout requires two genetic components working together:
Floxed (Conditional) Allele
The target gene contains LoxP sites flanking critical exons. The floxed allele functions normally until exposed to Cre recombinase. ingenious targeting laboratory can generate these conditional alleles specific to your research.
Inducible Cre Driver
A transgenic or knockin line expressing an inducible form of Cre recombinase. Cre activity is suppressed until an inducing agent (tamoxifen or doxycycline) is administered. There are repositories that offer these inducible lines. However, if you need a specialized inducible Cre driver, ingenious targeting laboratory has the expertise to produce these for you too.
When both components are combined in the same animal, gene deletion can be triggered at the time of the investigator's choosing by administering the inducing agent.
Tamoxifen Inducible (CreERT2) System
CreERT2 is the most widely used inducible Cre system. It consists of Cre recombinase fused to a modified estrogen receptor ligand binding domain:
- 1In the absence of tamoxifen, CreERT2 is sequestered in the cytoplasm
- 2Tamoxifen (or its metabolite 4 hydroxytamoxifen) binds the modified estrogen receptor
- 3Ligand binding triggers nuclear translocation of CreERT2
- 4Nuclear Cre catalyzes recombination between LoxP sites
- 5Gene deletion is permanent once recombination occurs
Recombination typically occurs within days of tamoxifen administration, with maximal deletion achieved by 1 to 2 weeks post treatment depending on dosing regimen.
Ubiquitous Inducible Cre
Ubiquitous CreERT2 drivers enable global gene deletion in adult animals:
Ubiquitous drivers provide adult onset global knockout, bypassing developmental lethality while achieving widespread gene deletion.
Tissue Specific Inducible Cre
Tissue specific CreERT2 drivers combine spatial and temporal control:
Tissue specific CreERT2 lines enable gene deletion in defined adult cell populations without affecting development of those tissues.
When to Use Inducible Systems
Bypassing Developmental Lethality
Many genes are essential for embryonic development. Conventional knockout is lethal, and even tissue specific conditional knockout may cause developmental defects if deletion occurs during organogenesis. Inducible systems enable gene deletion after development is complete, allowing study of adult gene function.
Distinguishing Developmental from Adult Roles
Genes may have distinct functions during development versus adulthood. Constitutive deletion conflates these roles. Inducible deletion in adults reveals the gene's ongoing function independent of its developmental requirements.
Example: A gene required for neuronal migration during development may also regulate synaptic plasticity in adults. Inducible knockout allows study of the adult synaptic function without the confounding effects of abnormal neuronal positioning.
Modeling Drug Target Inhibition
Inducible knockout most closely models therapeutic target inhibition: Patient has normal target function before treatment, drug administration causes acute target inhibition. Inducible knockout mirrors this: normal function until tamoxifen triggers deletion.
This makes inducible systems valuable for drug target validation, predicting consequences of pharmacological inhibition rather than lifelong gene absence.
Studying Acute vs Chronic Phenotypes
Constitutive knockouts represent chronic, lifelong gene loss. Inducible knockouts enable study of acute effects immediately following gene deletion, as well as progression of phenotypes over time. This temporal resolution is valuable for understanding disease mechanisms and therapeutic windows.
Avoiding Developmental Compensation
When genes are deleted early in development, compensatory mechanisms may mask the full phenotype. Related genes may upregulate, alternative pathways may activate. Deleting genes in adults, after these developmental adaptations have occurred, can reveal stronger or different phenotypes.
Technical Considerations
Tamoxifen Administration
Tamoxifen dosing affects recombination efficiency:
Higher doses and longer treatment increase recombination efficiency but may cause tamoxifen related effects. Optimize dosing for your specific CreERT2 line and target tissue.
Recombination Efficiency
Not all cells in a target population may undergo recombination:
- Efficiency varies by CreERT2 driver and target tissue
- Some cells may escape recombination
- Mosaic deletion may occur within tissues
- Validate recombination efficiency with reporter crosses
Cross inducible Cre to a reporter line (Rosa26 tdTomato) and assess reporter expression after tamoxifen to validate your specific experimental system.
Controls for Inducible Experiments
Proper controls account for tamoxifen effects and Cre expression:
- Floxed only + tamoxifen: Controls for tamoxifen effects
- CreERT2 only + tamoxifen: Controls for Cre expression and tamoxifen
- Floxed + CreERT2 without tamoxifen: Controls for leaky Cre activity
- Floxed + CreERT2 + tamoxifen: Experimental group
Leaky Cre Activity
Some CreERT2 lines show low level activity without tamoxifen (leaky expression). This can cause background recombination that accumulates over time. Evaluate potential leaky activity by assessing recombination in uninduced animals, particularly for long term studies.
Doxycycline Inducible (Tet) Systems
Tetracycline regulated systems use doxycycline to control gene expression:
Unlike CreERT2 where deletion is permanent, Tet systems controlling Cre expression can provide ongoing control. However, once recombination occurs at a locus, that deletion is irreversible regardless of subsequent doxycycline manipulation.
Selected Publications
Cre models generated by ingenious targeting laboratory:
Lee B, Kwon JT, Jeong Y, Caris H, Oh D, Feng M, Davila Mejia I, Zhang X, Ishikawa T, Watson BR, Moffitt JR, Chung K, Huh JR, Choi GB. 2025. Inflammatory and anti-inflammatory cytokines bidirectionally modulate amygdala circuits regulating anxiety. Cell 8(188): 2190-2202.e15.
Tsvilovskyy V, Ottenheijm R, Kriebs U, Schütz A, Diakopoulos KN, Jha A, Bildl W, Wirth A, Böck J, Jaślan D, Ferro I, Taberner FJ, Kalinina O, et al. 2024. OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage. J Clin Invest 134(7): e169428.
Souza G, Stornetta DS, Shi Y, Lim E, Berry FE, Bayliss DA, Abbott SBG. 2023. Neuromedin B-expressing neurons in the retrotrapezoid nucleus regulate respiratory homeostasis and promote stable breathing in adult mice. J Neurosci 43(30): 5501-5520.
What Researchers Say
“The people at InGenious are friendly, professional, and extremely good at what they do. I have made 5 Knockin mice with them and everything has gone like clockwork.”
— David B. Roth, MD, PhD
Perelman School of Medicine, University of Pennsylvania
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