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.
Highlights
Tamoxifen (TAM)-inducible Cre/loxP is one of the most widely used inducible systems for gene regulation
We noted a significant increase in the frequency of YFP positive CD45+ cells when treating mice with 2.4 mg TAM, reaching 40% by day 30 under the best conditions (Fig. 1B)
These data indicate that induction of Cre activity by TAM is dose-dependent and is enhanced when delivered IP
Summary
Tamoxifen (TAM)-inducible Cre/loxP is one of the most widely used inducible systems for gene regulation. Cre/ER recombinases remain unexpressed until activated by the TAM metabolite, which provides external and time-specific control of Cre activity This system is used to address the function of specific g enes[3,9,10,11], including those relating to immune cells. Reporter lines that are most widely utilized contain a fluorescent protein (e.g. YFP) in the ROSA26 locus to verify efficient expression in all cell types, including their developmental stages[13,14] These lines are mainly used for lineage tracing, taking advantage of permanent induction of the reporter even after TAM delivery is discontinued. Several studies have used TAM to decipher the functional relevance of conditional gene deletion in specific immune cell subsets through temporal control of Cre expression. We aimed to determine which combination of dosage and route of TAM administration achieves the most effective induction of Cre based on YFP reporter expression. TAM concentration in various organs and YFP induction rate in different immune cell populations were measured
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