Project description:We have generated a miR-17~92 fl/fl allele whose expression can be turned off conditionally by Cre recombinase in the miR-106a-363-/-;miR-106b-25-/- background. The mice were crossed to CD19-Cre mice to turn off the expression of miR-17~92 specifically in B cells and stimulated LPS/IL-4 for 13.5hr. Follicular B(FoB) cells were purified from CD19-Cre;miR-17~92 fl/fl;miR-106a-363;miR-106b~25 mice (TKO1, TKO2, TKO3) and WT (WT1, WTl2, WT3) by MACS depletion of cells positive for CD9, CD43, and CD93 (also known as AA4.1). The purified B cells were stimulated with LPS/IL-4 for 13.5hr in B cell media. The purity of follicular B cells was examined by flow cytometry and was greater than 95% for all samples. Total RNA was extracted using RNeasy kit (QIAGEN).
Project description:We have generated a miR-17-92 transgenic allele (termed miR-17-92 Tg) whose expression can be turned on conditionally by Cre recombinase (Xiao et al, Nature Immunology, 9:405-14, 2008). The mice were crossed to CD19-Cre mice to turn on the transgene expression specifically in B cells. Follicular B(FoB) cells were purified from CD19-Cre;miR-17~92 Tg/Tg mice (TG1, TG2, TG3) and miR-17~92 Tg/Tg mice (WT1, WT2, WT3) by MACS depletion of cells positive for CD5, CD43, and CD93 (also known as AA4.1). The purified B cells were stimulated with LPS/IL-4 for 13.5hr or 25.5hr. The purity of follicular B cells was examined by flow cytometry and was greater than 95% for all samples. Total RNA was extracted using RNeasy kit (QIAGEN).
Project description:We have generated a miR-17~92 transgene and fl/fl allele whose expression can be turned on and off conditionally by Cre recombinase. The mice were crossed to CD19-Cre mice to turn on and off the expression of miR-17~92 specifically in B cells and stimulated LPS/IL-4 for 25.5hr. tKO mice were generated in the miR-106a-363-/-;miR-106b-25-/- background, so that all three homologous cluster of miR-17~92 family miRNAs are deleted in B cells.
Project description:We have generated a miR-17-92 transgenic allele (termed miR-17-92 Tg) whose expression can be turned on conditionally by Cre recombinase (Xiao et al, Nature Immunology, 9:405-14, 2008). The mice were crossed to CD19-Cre mice to turn on the transgene expression specifically in B cells. Follicular B(FoB) cells were purified from CD19-Cre;miR-17~92 Tg/Tg mice (FoBtg1, FoBtg2, FoBtg3) and miR-17~92 Tg/Tg mice (FoBwt1, FoBwt2, FoBwt3) by MACS depletion of cells positive for CD5, CD43, and CD93 (also known as AA4.1). The purity of follicular B cells was examined by flow cytometry and was greater than 95% for all samples. Total RNA was extracted using RNeasy kit (QIAGEN).
