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Control of differentiated or undifferentiated state of ES cells with femto-injection method

机译:毫微微注射法控制ES细胞的分化或未分化状态

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摘要

Mouse embryonic stem (ES) cells are self-renewal and differentiation into derivatives of 3 primary germ layers. The mechanisms involved in maintaining the undifferentiated state of embryonic stem cells have not been shown clearly. In this study, we try to find "trigger genes" that determine fate of ES cells with femto-injection method. A glass capillary was heated and pulled using a laser puller (Model 2000, Sutter Instrument Co., Novato, CA, USA). The capillary was filled with a solution containing p CAG-Oct3/4-IRES-EGFP and connected to a capillary holder of the microinjector. Then the microinjector was attached on the three-dimensional micromanipulator of SMSR. After femto-injection of a plasmid solution into ES cells, the culture dish was incubated at 37℃. At 24h later, the cells were observed with a confocal laser scanning microscope (LSM510, Carl Zeiss Co., Ltd.) to acquire of EGFP fluorescent (Ex/Em = 488/507 nm) and morphological analysis. As a result, 10 %-injected cells were expressed EGFP fluorescent and some of them showed fiber-like protrusions.
机译:小鼠胚胎干(ES)细胞是自我更新的,并分化为3个主要细菌层的衍生物。维持胚胎干细胞未分化状态的机制尚未清楚显示。在这项研究中,我们试图找到通过飞针注射法确定ES细胞命运的“触发基因”。加热玻璃毛细管,并使用激光拉拔器(2000型,萨特仪器公司,加利福尼亚州诺瓦托,美国)拉动毛细管。用包含p CAG-Oct3 / 4-IRES-EGFP的溶液填充毛细管,并连接到微注射器的毛细管支架上。然后将显微注射器连接到SMSR的三维显微操纵器上。将质粒溶液毫微注射入ES细胞后,将培养皿在37℃下孵育。 24小时后,用共聚焦激光扫描显微镜(LSM510,Carl Zeiss Co.,Ltd。)观察细胞,以获取EGFP荧光(Ex / Em = 488 / 507nm)并进行形态分析。结果,注射了10%的细胞表达了EGFP荧光,并且其中一些显示了纤维状突起。

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