IMPROVED LENTIVIRAL VECTORS FOR PRIMATE EMBRYONIC STEM CELLS

改进的灵长类胚胎干细胞慢病毒载体

• 批准号: 7716406
• 负责人: James Alexander Thomson
• 金额: $ 8.19万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-23 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7716406
• 关键词: Back; Cells; Cloning; Computer Retrieval of Information on Scientific Projects Database; Funding; Genes; Genetic Markers; Grant; Institution; Kanamycin Kinase; Lentivirus Vector; Pharmaceutical Preparations; Primates; Protocols documentation; Puromycin; Research; Research Personnel; Resources; Role; Services; Site; Skin; Source; Stem cells; System; Testing; Transferase; United States National Institutes of Health; Viral; embryonic stem cell; expression vector; human embryonic stem cell; human embryonic stem cell line; improved; particle; pluripotency; promoter; self-renewal; size; transgene expression; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To generate lentiviral particles with improved long term site dependent expression in primate ES cells and their derivatives and to test specific genetic markers for their role in self renewal. In order to efficiently deliver exogenous genes into human embryonic stem cells (hESCs), we've optimized lentiviral expression system: 1) we decreased the vector size to improve the cloning of various genes; 2) we developed different expression vectors with different promoters such as EF1a promoter and PGK promoter to allow different levels of transgene expression, along with different drug-selection markers such as neomycin phosphotransferase and puromycin-N-acetyl-transferase; 3) We optimized lentiviral packaging protocols, allowing us to obtain viral particles with consistent high titers. We have successfully produced 14 new lentiviral constructs using genes found to be essential for pluripotency in ES cells. We have also produced lentiviral particles from these 14 new constructs. Using these lentiviral particles, we were able to successfully reprogram differentiated skin cells back to ES-like cells. The new cells are called ips cells. This research used WNPRC Stem Cell Resources, IS services and federally approved human ES cell lines.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 在灵长类动物ES细胞及其衍生物中产生具有改善的长期位点依赖性表达的慢病毒颗粒，并测试特定的遗传标志物在自我更新中的作用。 为了有效地将外源基因传递到人类胚胎干细胞（HESC）中，我们优化了慢病毒表达系统：1）我们降低了载体的大小以改善各种基因的克隆； 2）我们与不同的启动子（例如EF1A启动子和PGK启动子）开发了不同的表达载体，以允许不同水平的转基因表达，以及不同的药物选择标记物，例如neomycin磷酸转移酶和puro霉素-N-乙酰基转移酶； 3）我们优化了慢病毒包装方案，使我们能够获得具有一致的高滴度的病毒颗粒。我们使用发现对ES细胞中多能性至关重要的基因成功生产了14种新的慢病毒构建体。我们还从这14种新结构中产生了慢病毒颗粒。使用这些慢病毒颗粒，我们能够成功地将分化的皮肤细胞重新编程回ES样细胞。新细胞称为IPS细胞。这项研究使用了WNPRC干细胞资源，是服务和联邦批准的人类ES细胞系。