Figure 1.
Development of miRNA rescue screen in mature B cells stimulated to CSR to IgG1 implicates miR-222 as a regulator. (A) Timing and stimulation conditions to induce Cγ1-Cre deletion of Dgcr8 and transfect miRNA and analyze CSR by flow cytometry. (B) Representative flow plot of gating strategy for YFP reporter and IgG1 surface expression at day 2 during activation. (C) RT-qPCR of D8-WT, D8-HET, D8-KO B cell Dgcr8 mRNA and miRNA expression throughout culturing (Dgcr8 normalized to Gapdh, miRNAs normalized to small nuclear RNA U6). Data are the average of two mice from each genotype. (D) Screen results from the individual miRNA rescue transfections performed at day 0 and read out by flow cytometry at the day 2 time point. Z-score was calculated by z = (x1 − xn)/σ, where x1 is the specific IgG1 percentage, xn is the mean of all surface IgG1 percentages, and σ is the SD. Control bar is the average of six transfections ranging from +1 to −1 Z-score averaged.

Development of miRNA rescue screen in mature B cells stimulated to CSR to IgG1 implicates miR-222 as a regulator. (A) Timing and stimulation conditions to induce Cγ1-Cre deletion of Dgcr8 and transfect miRNA and analyze CSR by flow cytometry. (B) Representative flow plot of gating strategy for YFP reporter and IgG1 surface expression at day 2 during activation. (C) RT-qPCR of D8-WT, D8-HET, D8-KO B cell Dgcr8 mRNA and miRNA expression throughout culturing (Dgcr8 normalized to Gapdh, miRNAs normalized to small nuclear RNA U6). Data are the average of two mice from each genotype. (D) Screen results from the individual miRNA rescue transfections performed at day 0 and read out by flow cytometry at the day 2 time point. Z-score was calculated by z = (x1 − xn)/σ, where x1 is the specific IgG1 percentage, xn is the mean of all surface IgG1 percentages, and σ is the SD. Control bar is the average of six transfections ranging from +1 to −1 Z-score averaged.

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