Generation of CD4CreER(T²) transgenic mice to study development of peripheral CD4-T-cells

Abstract

After thymic emigration CD4-T-cells continue to differentiate into multiple effector and suppressor sublineages in peripheral lymphoid organs. In vivo analysis of peripheral CD4-T-cell differentiation has relied on animal models with targeted gene mutations. These are expressed either constitutively or conditionally after Cre mediated recombination. Available Cre transgenic strains to specifically target T-cells act at stages of thymocyte development that precede thymic selection. Tracing gene functions in CD4-T-cell development after thymic exit becomes complicated when the targeted gene is essential during thymic development. Other approaches to conditionally modify gene functions in peripheral T-cells involve infection of in vitro activated cells with Cre expressing lenti-, retro-, or adenoviruses, which precludes in vivo analyses. To study molecular mechanisms of peripheral CD4-T-cell differentiation in vivo and in vitro we generated transgenic mice expressing a tamoxifen inducible Cre recombinase (CreER(T2) ) under the control of the CD4 gene promoter. We show here that in CD4CreER(T2) mice Cre is inducibly and selectively activated in CD4-T-cells. Tamoxifen treatment both in vivo and in vitro results in efficient recombination of loci marked by LoxP sites. Moreover, this strain shows no abnormalities related to transgene insertion. Therefore it provides a valuable tool for studying gene function during differentiation of naïve peripheral CD4-T-cells into effector or suppressor sub-lineages.

Figure 1. Generation of CD4-CreER^T2 mice

(a) Schematic diagram of the construct used for the generation of the CD4CreER^T2 transgenic mouse strain. Colored boxes show the Cd4 gene promoter/enhancer/silencer the Cre the ER^T2 moiety and the HGH polyadenylation as indicated. Restriction sites and sizes of the fragments used for the cloning are shown (see Methods). (b) Representative genotyping PCR analysis of 4 weeks old CD4CreER^T2/R26R-EYFP double transgenic mice. A negative control (-ve) is also shown.

Figure 2. In-vitro activation in CD4CreER^T2 in CD4⁺ T cells

(a) Lymphocytes isolated from spleen and lymph nodes of the indicated mice were cultured for 3 days in medium containing the indicated concentrations of 4-OHT. Treated cells were stained for surface expression of CD4 and analyzed by FACS. Line histograms show representative EYFP expression in gated CD4 T-cells for the indicated treatment and mouse strain. (b) Histogram bars show the average frequency of EYFP+ CD4 T-cells after 4-OHT treatment of lymphocytes isolated from 4 independent mice for each indicated strain and 4-OHT concentration p< 0.0001. Results are representative of two independent experiments.

Figure 3. In-vivo Cre activation

The indicated mice were treated in-vivo with tamoxifen (See Methods). EYFP expression was analyzed as a measure of Cre activation and representative FACS plots of the indicated parameters are shown. In (a) histogram plots show EYFP expression in un-gated live cells from the spleen of the indicated mice. Tow parameter contour plots show CD4 versus CD8 expression in gated YFP+ and YFP− cells as shown by the arrows. (b) Line histograms show EYFP expression in cells harvested from the spleen of the indicated mice and gated for surface expression of the indicated markers. Data are representative of three independent experiments.

Figure 4. Stabilization of β-catenin in CD4 T-cells correlates with YFP expression

CD4+ T cells from the spleen and lymph nodes of CD4CreER^T2/R26R-EYFP/ Ctnnb1^ex3 compound mutant mice were treated with 0.5 μM 4-OHT. (a) Histogram plots show EYFP expression for the indicated length of treatment. EYFP+ and EYFP− cells were sorted on day 3 of treatment (b) Shows Western Blot analysis for β-catenin in lysates from the indicated sorted populations of CD4-T-cells. Data show cells isolated from two independent mice. Actin is shown as a loading control. One of 2 independent experiments is shown.