Optimization of tamoxifen-induced Cre activity and its effect on immune cell populations
Rachel S Donocoff1, Nato Teteloshvili2, Hyunsoo Chung2
1Institute of Comparative Medicine, Columbia University Medical Center, Columbia University, New York, NY, 10032, USA.
Abstract:
Tamoxifen (TAM) inducible Cre recombinase system is an essential tool to study gene function when early ablation or overexpression can cause developmental defects or embryonic lethality. However, there remains a lack of consensus on the optimal route and dosage of TAM administration in vivo. Here, we assessed dosage and delivery of TAM for activation of Cre in immune cell subsets assessed longitudinally and spatially using transgenic mice with ubiquitously expressed Cre/ER and the Cre-inducible fluorescent reporter YFP. After comparing two TAM delivery methods (intraperitoneal versus oral gavage) and different doses, we found that 3 mg of TAM administered orally for five consecutive days provides maximal reporter induction with minimal adverse effects in vivo. Serum levels of TAM peaked 1 week after initiating treatment then slowly decreased, regardless of dosing and delivery methods. TAM concentration in specific tissues (liver, spleen, lymph nodes, and thymus) was also dependent on delivery method and dose. Cre induction was highest in myeloid cells and B cells and substantially lower in T cells, and double-positive thymocytes had a notably higher response to TAM. In addition to establishing optimal dose and administration of TAM, our study reveals a disparate activity of Cre in different cell immune populations when using Cre/ER models.
Insights
Optimal tamoxifen (TAM) administration for Cre-ER activation in mice involves 3 mg orally for five days, yielding maximal reporter induction with minimal side effects. This method ensures effective gene function studies in immune cells.
Area of Science:
- Immunology
- Genetics
- Pharmacology
Background:
- The tamoxifen (TAM)-inducible Cre recombinase system is crucial for studying gene function, especially when developmental issues arise from early gene manipulation.
- Optimal protocols for TAM administration (dosage and delivery route) are not standardized, hindering reproducible research.
Purpose of the Study:
- To determine the optimal dosage and delivery method of tamoxifen for Cre-ER activation in immune cell subsets.
- To assess the longitudinal and spatial distribution of tamoxifen and its effects on Cre induction across different immune populations.
Main Methods:
- Utilized transgenic mice with Cre/ER and a Cre-inducible YFP reporter.
- Compared intraperitoneal injection versus oral gavage for TAM delivery.
- Evaluated various TAM doses and assessed YFP reporter expression in immune cells over time.
Main Results:
- Oral administration of 3 mg of TAM for five consecutive days resulted in maximal YFP reporter induction with minimal adverse effects.
- Serum TAM levels peaked at 1 week post-treatment and declined slowly, irrespective of dose or delivery method.
- TAM tissue concentration varied by delivery method and dose; Cre induction was highest in myeloid and B cells, lower in T cells, with notable response in double-positive thymocytes.
Conclusions:
- Established an optimal protocol (3 mg oral TAM for 5 days) for tamoxifen-inducible Cre-ER system activation in mice.
- Demonstrated differential Cre activity across immune cell populations, highlighting the importance of considering cell-specific responses in experimental design.
