Co-Regulation of miR-133b and miR-206 in the Il17a/f Locus Figure 7. Functional outcome of ectopically expressed miR-133b and miR-206 in vitro and in vivo. (A) In vitro assay. MACS-enriched CD4+ T cells from TCR-transgenic DO.11.10 mice were retrovirally transduced with miR-133b or miR-206 or with both and stimulated under Th17 polarization inducing conditions. Frequency of IL-17 producing CD4+ T cells among transduced (GFP+) and non-transduced (GFP 2 ) cells of the same well was compared. One representative of two independent experiments with similar results is shown. (B, C) Frequency of IL-17 producing cells overexpressing miR-133b or miR-206 in vivo. Lineage negative bone marrow was transduced with miR-133b or miR-206 or with the empty vector MDH1-PGK-GFP2.0 and served to reconstitute lethally irradiated C57BL/6 wild type mice. After 8–10 weeks, chimeras were analyzed for the occurrence and frequency of IL-17 producing cells within the transduced GFP+ and non-transduced GFP2 lymphocytes from peripheral lymph nodes and spleen. (B) Representative gating strategy after excluding autofluorescent and B220+ cells. (C) Frequency of IL-17 producing CD4+ and CD42 cells, respectively. At least 5 chimeric mice were individually analyzed for each condition. doi:10.1371/journal.pone.0020171.g007 PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20171