Faust N, Bonifer C, Wiles M V, Sippel A E
Institut für Biologie III, Universität Freiburg, Germany.
DNA Cell Biol. 1994 Sep;13(9):901-7. doi: 10.1089/dna.1994.13.901.
In an effort to study basic principles of marker gene activation during myeloid lineage development, we established an in vitro differentiation system for macrophages based on mouse embryonic stem (ES) cells. Under the influence of defined cytokines, ES cells gave rise to a cell population consisting predominantly of macrophages. We could show, that expression of the mouse lysozyme M gene is a faithful internal standard for indicating the proportion of macrophage cells in the differentiation culture. This controlled in vitro differentiation system can be used for quantitative studies on transgene activation. Undifferentiated ES cells were stably transfected with a construct carrying the chicken lysozyme gene locus, which had been shown previously to express lysozyme RNA cell type specifically and position independently in macrophages of transgenic mice. In undifferentiated transfected ES cell clones, the transgene was consistently inactive. Upon in vitro differentiation, the transgene was expressed exclusively in macrophages and its level of activity was independent of the chromosomal site of integration. The in vitro cell differentiation system presented here will be useful to study the cis- and trans-regulatory requirements of myeloid-specific gene activation and the influence of hematopoietic regulators on myelopoiesis through their effect on transfected marker gene expression.
为了研究髓系谱系发育过程中标记基因激活的基本原理，我们基于小鼠胚胎干细胞建立了一种巨噬细胞体外分化系统。在特定细胞因子的影响下，胚胎干细胞产生了一个主要由巨噬细胞组成的细胞群体。我们发现，小鼠溶菌酶M基因的表达是一个可靠的内参标准，可用于指示分化培养物中巨噬细胞的比例。这个可控的体外分化系统可用于转基因激活的定量研究。将携带鸡溶菌酶基因座的构建体稳定转染未分化的胚胎干细胞，该构建体先前已被证明在转基因小鼠的巨噬细胞中可细胞类型特异性且位置独立地表达溶菌酶RNA。在未分化的转染胚胎干细胞克隆中，转基因始终处于无活性状态。体外分化后，转基因仅在巨噬细胞中表达，其活性水平与整合的染色体位点无关。本文介绍的体外细胞分化系统将有助于研究髓系特异性基因激活的顺式和反式调控需求，以及造血调节因子通过对转染标记基因表达的影响对髓系造血的作用。
