Regulatable In Vivo Biotinylation Expression System in Mouse Embryonic Stem Cells

Embryonic stem (ES) cells have several unique attributes, the two most important of which are they can differentiate into all cell types in the body and they can proliferate indefinitely. To study the regulation of these phenomena, we developed a regulatable in vivo biotinylation expression system in mouse ES cells. The E. coli biotin ligase gene BirA, whose protein product can biotinylate a 15-aa peptide sequence, called the AviTag, was cloned downstream of an IRES. The primary vector containing the doxycycline controlled transactivator gene tTA and IRES-BirA was knocked into the ROSA26 locus by homologous recombination. The secondary vector containing the AviTag tagged hKlf4 gene was exchanged into the ROSA26 locus using Cre recombinase. Western blot analysis showed that the doxycycline induced BirA protein can biotinylate the doxycycline induced AviTag tagged hKlf4 protein. The induction of hKlf4 repressed cell growth in the presence or absence of LIF. Chromatin immunoprecipitation assays using streptavidin beads showed that the AviTag tagged hKlf4 protein could enrich the Nanog enhancer. Our results suggested that the regulatable biotinylation system is promising for the gene function studies in mouse ES cells.胚胎干细胞（Embryonic Stem Cells, ES）具有多项独特特性，其中最为关键的两项是可分化为机体所有细胞类型，且能无限增殖。为研究这两类现象的调控机制，我们在小鼠ES细胞中构建了可调控的体内生物素化表达系统。大肠杆菌（Escherichia coli, E. coli）的生物素连接酶基因BirA，其编码的蛋白可对一段名为AviTag的15-氨基酸（15-aa）肽段进行生物素化修饰，该基因被克隆至内部核糖体进入位点（Internal Ribosome Entry Site, IRES）的下游区域。我们将携带多西环素（Doxycycline）调控的反式激活因子基因tTA与IRES-BirA元件的初始载体，通过同源重组整合至ROSA26基因座。随后，携带AviTag标签化hKlf4基因的次级载体，通过Cre重组酶（Cre recombinase）介导的重组交换整合至ROSA26基因座。蛋白质免疫印迹（Western Blot, WB）分析结果显示，多西环素诱导表达的BirA蛋白，可对多西环素诱导的AviTag标签化hKlf4蛋白进行生物素化修饰。诱导表达hKlf4可在有无白血病抑制因子（Leukemia Inhibitory Factor, LIF）的培养条件下抑制细胞增殖。采用链霉亲和素磁珠的染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）实验结果显示，AviTag标签化hKlf4蛋白可富集Nanog增强子区域。本研究结果表明，该可调控生物素化系统在小鼠ES细胞的基因功能研究中具有良好的应用前景。