Use of Cre-adenovirus and CAR transgenic mice for efficient deletion of genes in post-thymic T cells

Abstract

Conditional gene deletion using lineage-specific transgenic expression of Cre has been useful for defining the role of specific gene products in mice in vivo. However, this technology has had limitations for studies of peripheral T cell biology, since the T-lineage promoters commonly used are active early in thymic development. As such, T cell development can be altered by the resulting genetic alterations, thus limiting the interpretation of the data in post-thymic T cell studies. Thus, new strategies are needed to delete targeted genes directly in peripheral T lymphocytes. The availability of transgenic mice expressing the CAR in the T cell compartment enabled testing of Cre-mediated recombination using an adenoviral vector in naïve peripheral T cells in vitro, even without cellular activation. Using Rosa26R reporterxCAR transgenic mice, we describe conditions by which Cre-mediated deletion of targeted genes can be achieved with primary T cells in vitro. These cells can also be adoptively transferred into defined recipient mice for study in vivo. We use conditional PTEN-deficient mice as proof of concept to confirm the value of this technique for deleting a negative regulator of T cell activation. This technology should be broadly applicable for studies of T cell-specific gene deletion to gain understanding of function in the post-thymic T cell compartment.

Fig. 1

Adenoviral-mediated Cre expression in CAR Tg T cells. (a) Western blot analysis of cultured CAR Tg CD4⁺ Th1 T cell clone transduced with either EV or Adeno-Cre. (b) Western blotting of splenic T cells isolated from CAR Tg mice transduced with EV or Adeno-Cre. Similar results were observed in at least two independent experiments.

Fig. 2

Cre-mediated deletion is achieved in primed T cells after adeno-Cre transfection. (a) Freshly isolated T cells from CAR Tg×Rosa26R reporter mice were transduced with either EV or Adeno-Cre and incubated overnight. Transduced cells were then primed in culture for 3 days, and β-gal activity was analyzed by flow cytometry. (b) The same T cells were separated and primed in culture for 3 days, then transduced with either EV or Adeno-Cre, and β-gal activity was analyzed by flow cytometry on the next day. (c) T cells from CAR Tg×Rosa26R reporter mice were isolated and transduced, and β-gal activity was analyzed by flow cytometry on the next day. Similar results were seen in at least two experiments.

Fig. 3

Cre-mediated deletion is not cell cycle dependant. (a) CAR Tg×Rosa26R reporter T cells were isolated, transduced, and primed in culture for 3 days with or without the presence of the cell cycle blocker, aphidicolin. T cell proliferation was measured by thymidine incorporation. (b) CAR Tg×Rosa26R reporter were treated as in (a) and flow cytometry was performed to analyze β-gal activity.

Fig. 4

Extended cell culture period is needed for Cre-mediated deletion. Freshly isolated CAR Tg×Rosa26R reporter T cells were transduced with either EV or Adeno-Cre and incubated overnight. Transduced T cells were maintained in culture for 3 days in the presence of 1 ng/ml IL-7, and β-gal activity was analyzed by flow cytometry.

Fig. 5

Cre-mediated deletion is maintained in vivo. CAR Tg×Rosa26R reporter T cells were isolated, transduced, and maintained in culture for 3 days at the presence of IL-7. (a) β-gal activity was assessed by flow cytometry prior to in vivo transfer. (b) Transduced T cells were adoptively transferred into P14 TCR Tg RAG1^−/− mice intravenously. One week late, T cells were purified and β-gal activity was analyzed by flow cytometry.

Fig. 6

Cre-mediated deletion of PTEN expression in peripheral T cells. CAR Tg mice were intercrossed with conditional PTEN gene-targeted mice. Splenic T cells were isolated, transduced with either empty vector or adeno-Cre, and maintained in culture for 9 days in IL-7. (a) Western blot analysis was performed for Cre and PTEN expression. (b) Transduced cells were stimulated overnight with beads conjugated with anti-CD3 mAb (0.001 μg/ml) and anti-CD28 mAb (1 μg/ml) and IL-2 production was measured by ELISA. *, undetectable. (c) Western blots were scanned and densitometry was performed to determine the ratio of PTEN to ERK signal. (d) Th1 clones were derived from CAR/PTEN-flox mice and adeno-Cre-mediated deletion was performed as for primary T cells, with culture for 14 days prior to Western blot analysis. Similar results were observed in three independent experiments.