Gropp Michal, Reubinoff Benjamin E
The Hadassah Human Embryonic Stem Cell Research Center, Goldyne Savad Institute of Gene Therapy, Hadassah University Medical Center, Jerusalem, Israel.
Cloning Stem Cells. 2007 Fall;9(3):339-45. doi: 10.1089/clo.2006.0077.
Genetic modifications of human embryonic stem cells (hESCs) that will efficiently promote stable homogenous gene silencing, and will also allow monitoring of the silencing level, may be invaluable for the study of function of genes in early human embryogenesis, differentiation, and maintenance of pluripotency of hESCs. RNA-mediated interference (RNAi) emerges as a highly efficient tool for specific knockdown of gene expression. Lentiviruses are efficient vectors for the delivery and stable expression of transgenes in hESCs. We sought to develop a lentiviral-RNAi-based system that will efficiently induce homogenous gene silencing and will allow the monitoring of its relative level in hESCs. Dual-promoter lentiviral vectors coexpressing an RNAi cassette and a reporter gene were initially used for efficient and stable induction of heterogeneous levels of gene silencing in polyclonal hESCs. This step was further combined with the isolation of transduced clones with different homogenous levels of gene silencing. The level of silencing in each of the clones correlated and could be monitored by the level of expression of the vector's reporter transgene. Thus, our system allows easy identification of clones with relatively different homogenous levels of gene silencing. Our approach would be valuable for the study of function of genes, in particular those whose role in hESCs biology depends on their level of expression.
对人类胚胎干细胞（hESC）进行基因改造，以有效促进稳定的基因沉默，并能监测沉默水平，这对于研究人类早期胚胎发育、分化以及hESC多能性维持过程中基因的功能可能具有重要价值。RNA介导的干扰（RNAi）已成为特异性敲低基因表达的高效工具。慢病毒是在hESC中传递和稳定表达转基因的有效载体。我们试图开发一种基于慢病毒-RNAi的系统，该系统能有效诱导hESC中的基因沉默，并能监测其相对水平。最初，共表达RNAi盒和报告基因的双启动子慢病毒载体用于在多克隆hESC中高效稳定地诱导不同水平的基因沉默。这一步骤进一步与分离具有不同基因沉默水平的转导克隆相结合。每个克隆中的沉默水平相互关联，并且可以通过载体报告转基因的表达水平进行监测。因此，我们的系统能够轻松识别具有相对不同基因沉默水平的克隆。我们的方法对于研究基因功能，特别是那些在hESC生物学中的作用取决于其表达水平的基因，具有重要价值。
