ERT2-Cre诱导的Rosa26R-YFP报告基因系统标记小鼠造血干祖细胞的条件和效率检测

目的 探索ERT2-Cre诱导的黄色荧光蛋白（YFP）报告基因系统标记小鼠造血干祖细胞的条件和效率.方法 通过基因型分型的方法,从Rosa26R-YFP小鼠与ERT2-Cre小鼠杂交得到的后代中,筛选出YFP及ERT2-Cre双阳性转基因杂交小鼠;采用免疫磁珠法从双阳性转基因小鼠骨髓细胞中富集c-kit＋造血干祖细胞群进行体外培养,并用不同浓度4-羟基它莫西芬（OHT）作用不同时间以诱导YFP表达;流式细胞术检测c-kit＋造血干祖细胞中YFP的表达量.结果 Rosa26R-YFP小鼠与ERT2-Cre小鼠的杂交后代共9只,采用基因型分型方法筛选出双阳性转基因小鼠5只;体外培养的c-kit＋细胞部分形态发生变化,提示存在细胞分化;流式细胞术检测到c-kit＋细胞中YFP表达量可达13.7％.结论 YFP报告基因系统在OHT诱导的ERT2-Cre作用下,能有效标记小鼠造血干祖细胞.

Conditions and efficiency of Rosa26R-YFP reporter gene system labeled mouse hematopoietic stem and progenitor cells induced by ERT2-Cre

Objective To explore the conditions and efficiency of YFP-labeled mouse hematopoietic stem and progenitor cells induced by ERT2-Cre.Methods ERT2-Cre and YFP double-positive transgenic hybrid mice from all offsprings of Rosa-YFP mice and ERT2-Cre mice were obtained by genotyping,and c-kit＋ cells were enriched in vitro using magnetic activated cell sorting from bone marrow cells of double-positive transgenic hybrid mice.The expression of YFP was induced by different concentrations of 4-hydroxytamoxifen （OHT） with different periods,and the expressing efficiency of YFP in c-kit＋ cells was detected by flow cytometry.Results Five out of nine Rosa26R-YFP and ERT2-Cre mice crossbred progenies were YFP and Cre transgene double positive.Some morphological changes of c-kit＋ cells cultured in vitro suggested the presence of cell differentiation.The expressing efficiency of YFP in c-kit ＋ cells was up to 13.7％.Conclusions YFP reporter gene system can effectively mark mouse hematopoietic stem and progenitor cells induced by ERT2-Cre with the exist of OHT in vitro.