Microarray analysis to unravel the function of C-terminus domain of EBF1 by replacement approach. Mus musculus

To unravel the function of C-terminus domain of EBF1, a replacement microarray analysis was performed by transducing Ebf1wt and Ebf1DC expressing vectors intoA-MuLV transformed pro-B (Ebf1fl/fl;RERTCre) cells. Overall design: A-MuLV transformed Ebf1fl/fl RERTCre pro-B cells were treated with 2μM 4-hydroxy-tamoxifen (4-OHT) 24h after transduction. Five days after treatment, cells were sorted as GFP+. More than two week later, the replacement was validated by IB and the cells were used for downstream analyses. As control, mock-transduced cells were harvested four days after tamoxifen treatment before cell death was observed.

为阐明EBF1（early B-cell factor 1）C端结构域的功能，本研究通过将携带Ebf1野生型（Ebf1wt）与Ebf1DC的表达载体转导至A-MuLV（Abelson murine leukemia virus）转化的原B细胞（Ebf1fl/fl;RERTCre）中，开展了替换型微阵列分析。实验设计概况：转导24小时后，向A-MuLV转化的Ebf1fl/fl RERTCre原B细胞中加入2μM 4-羟基他莫昔芬（4-hydroxy-tamoxifen，简称4-OHT）进行处理；处理5天后，分选获取GFP阳性细胞。时隔两周以上后，通过免疫印迹（Immunoblotting，IB）验证了基因替换效果，并将该细胞用于后续实验分析。作为对照，在他莫昔芬处理后4天、细胞尚未出现死亡时，收集空载转导的细胞。