Cheng T, Shen H, Giokas D, Gere J, Tenen D G, Scadden D T
MGH Cancer Center, Charlestown 02129, USA.
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13158-63. doi: 10.1073/pnas.93.23.13158.
A hierarchical order of gene expression has been proposed to control developmental events in hematopoiesis, but direct demonstration of the temporal relationships between regulatory gene expression and differentiation has been difficult to achieve. We modified a single-cell PCR method to detect 2-fold changes in mRNA copies per cell (dynamic range, 250-250,000 copies/cell) and used it to sequentially quantitate gene expression levels as single primitive (CD34+,CD38-) progenitor cells underwent differentiation to become erythrocytes, granulocytes, or monocyte/macrophages. Markers of differentiation such as CD34 or cytokine receptor mRNAs and transcription factors associated with their regulation were assessed. All transcription factors tested were expressed in multipotent progenitors. During lineage-specific differentiation, however, distinct patterns of expression emerged. SCL, GATA-2, and GATA-1 expression sequentially extinguished during erythroid differentiation. PU.1, AML1B, and C/EBP alpha expression profiles and their relationship to cytokine receptor expression in maturing granulocytes could be distinguished from similar profiles in monocytic cells. These data characterize the dynamics of gene expression accompanying blood cell development and define a signature gene expression pattern for specific stages of hematopoietic differentiation.
有人提出基因表达的层级顺序可控制造血过程中的发育事件，但要直接证明调控基因表达与分化之间的时间关系却很难实现。我们改进了一种单细胞PCR方法，以检测每个细胞中mRNA拷贝数的2倍变化（动态范围为250 - 250,000拷贝/细胞），并在单个原始（CD34 +，CD38 -）祖细胞分化为红细胞、粒细胞或单核细胞/巨噬细胞的过程中，用该方法依次定量基因表达水平。我们评估了分化标志物，如CD34或细胞因子受体mRNA以及与其调控相关的转录因子。所有检测的转录因子均在多能祖细胞中表达。然而，在谱系特异性分化过程中，出现了不同的表达模式。在红系分化过程中，SCL、GATA - 2和GATA - 1的表达依次消失。在成熟粒细胞中，PU.1、AML1B和C/EBPα的表达谱及其与细胞因子受体表达的关系，可与单核细胞中的类似谱相区分。这些数据描绘了伴随血细胞发育的基因表达动态，并为造血分化的特定阶段定义了标志性基因表达模式。
