Animal work was performed according the Animals (Scientific Procedures) Act, UK. Dicerlox/lox mice were crossed with LckCre transgenic mice (17 (link)) to generate lckCre DicerΔ/Δ mice. Thymocytes were stained, analyzed, and sorted by flow cytometry as described previously (24 (link)). Where indicated, thymocytes were incubated with 40 nM DiOC6 (Molecular Probes) for 10 min at 37°C as described previously (20 (link)). To down-regulate Tdt expression, DP thymocytes were cultured with 7.5 ng/ml PMA (Sigma-Aldrich) and 180 ng/ml ionomycin (Sigma-Aldrich) as described previously (28 (link)).
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Dicer Knockout Thymocyte Analysis
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Other organizations : Medical Research Council, Imperial College London, Howard Hughes Medical Institute, University of Washington, University of California, Los Angeles
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Protocol cited in 11 other protocols
Variable analysis
independent variables
- Cre recombinase expression (lckCre)
- Treatment with PMA and ionomycin
- Dicer expression (Dicer^Δ/Δ)
- Thymocyte staining, analysis, and sorting by flow cytometry
- Thymocyte incubation with DiOC6
- Tdt expression
- Animal species and strains (Dicer^lox/lox mice, LckCre transgenic mice)
- Experimental procedures (as described previously in references 17, 24, and 20)
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