Overview
Tamoxifen inducible Cre (CreERT2) is a temporal gene deletion system that fuses Cre recombinase to a mutant estrogen receptor ligand-binding domain (ERT2). This fusion protein remains sequestered in the cytoplasm by HSP90 chaperones until tamoxifen administration triggers nuclear translocation and LoxP recombination. This system enables gene deletion at defined timepoints in adult animals, bypassing embryonic lethality and avoiding developmental compensation mechanisms.
Mechanism of CreERT2 Activation
In the absence of ligand, the ERT2 domain binds HSP90 chaperone proteins, sequestering the CreERT2 fusion protein in the cytoplasm away from nuclear DNA. Tamoxifen (or its active metabolite 4-hydroxytamoxifen) binds to ERT2, displacing HSP90 and exposing a nuclear localization signal. CreERT2 translocates to the nucleus where Cre catalyzes recombination between LoxP sites, permanently deleting the floxed sequence. Recombination is heritable through subsequent cell divisions.
Tamoxifen Dosing and Administration
Common administration routes include intraperitoneal injection (75-100 mg/kg in corn oil, most common), oral gavage (similar dosing), or tamoxifen-containing chow (250-500 mg/kg diet for extended dosing). Typical protocols use 3-5 consecutive daily injections. Recombination efficiency varies by tissue and Cre driver line. Researchers should verify deletion efficiency in target tissues by PCR, qPCR, or immunoblotting.
Frequently Asked Questions
What are the advantages of CreERT2 over constitutive Cre?
CreERT2 enables temporal control, allowing gene deletion at defined timepoints after normal development. This bypasses embryonic lethality of essential genes, avoids developmental compensation that can mask adult phenotypes, enables comparison of early versus late gene deletion effects, and provides matched controls (same genotype with and without tamoxifen treatment).
How quickly does gene deletion occur after tamoxifen administration?
Recombination at the DNA level occurs within 24-72 hours after tamoxifen administration. However, protein turnover determines functional knockout kinetics. For stable proteins, complete depletion may take 7-14 days. For rapidly turning over proteins, functional effects can be observed within 2-3 days. Researchers should verify both DNA recombination and protein loss in target tissues.
Is there background Cre activity without tamoxifen?
Most CreERT2 systems show minimal leakiness (background activity without tamoxifen), but this varies by Cre driver line and tissue. Some CreERT2 lines show detectable background recombination (1-10%) in sensitive reporter assays. For critical experiments, include tamoxifen-only and Cre-only controls to quantify background activity. Second-generation ERT2 systems have reduced leakiness compared to original ER fusions.
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