180 EFFICIENT GENE SILENCING USING siRNA IN MOUSE AND MONKEY ES CELLS AND DIFFERENTIATION

Abstract

Gene silencing by RNA interference (RNAi) using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool for silencing specific genes in various organisms. As compared with transgene-based RNAi using shRNA expression vectors, chemically synthesized siRNAs have an advantage in that they do not modify genome organization. This nature is ideal as a differentiation method for embryonic stem (ES) cells. The objective of this work was to develop an efficient method to repress a specific gene expression in mouse and monkey ES cells using chemically synthesized siRNA, and to investigate whether this transient gene silencing can be used as a new differentiation method of ES cells. First, we tried to knock down the expression of enhanced green fluorescent protein (EGFP) gene in GFP-expressing mouse ES cells. We found that lipofection was effective to deliver siRNA into mouse ES cells. Suppression of EGFP expression was observed by fluorescence microscopy after 24 h of transfection and the silencing continued at least for 5 days. FACS analysis revealed that 86% of cells showed suppression of EGFP at 48 h after transfection. Then we tried to suppress endogenous gene Oct4 that plays an important role in the maintenance of pluripotency and the lineage commitment in mouse ES cells. We performed RT-PCR analysis and western blotting to assay for Oct4 mRNA and protein at 24, 48, 72, and 96 h after transfection. Hand1 and Cdx2, transcription factors implicated in trophoblast differentiation, were also analyzed. RT-PCR analysis showed a reduced level of Oct4 mRNA at 24–96 h. Reduction of Oct4 protein was confirmed by western blotting and the reduced level was still maintained at 96 h after transfection. RT-PCR analysis also showed up-regulation of Hand1 and Cdx2 concomitant with the suppression of Oct4. Furthermore, ES cells changed their morphology into a large and flattened shape that is characteristic of trophoblast cells. These results suggested that the transient suppression of Oct4 induced differentiation of mouse ES cells to trophectoderm cells as expected. Therefore our data imply that the chemically synthesized siRNA can be used to differentiate ES cells. Next, we tried to suppress EGFP and Oct4 in monkey ES cells. In monkey ES cells, we found that the Sendai virus (hemagglutinating virus of Japan, HVJ) envelope was suitable to deliver siRNA into cells. With this method, we detected efficient silencing of EGFP and Oct4 by fluorescence microscopy, RT-PCR analysis, and western blotting. In the case of monkey ES cells, however, no morphological change was observed by Oct4 suppression at 96 h after transfection. These results suggest possible diversity between murine and primate ES cells in the differentiation process. This work was supported by grants from the Ministry of Education, Science, Sports, and Culture (14580798, 13358014 and 14380382).