• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>BMC Neuroscience >c-MycER TAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain
【24h】

c-MycER TAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain

机译：植入MCAo啮齿动物大脑的转基因人类神经干细胞系中的c-MycER TAM转基因沉默

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Background The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro , 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo , CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively. Results The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence. Conclusion In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.
机译：背景技术人类神经干细胞系CTX0E03被开发用于基于细胞的慢性中风致残性治疗。 CTX0E03衍生自胎儿皮质脑组织，是一种克隆细胞系，其中包含通过逆转录病毒感染递送的c-mycER TAM 转基因的单个副本。在4-羟基他莫昔芬（4-OHT）的条件调节下，c-mycER TAM 使CTX0E03细胞能够大规模稳定储存。在这项研究中，我们调查了该转基因在体外的生长停滞（EGF，bFGF和4-OHT撤离）之后以及脑内植入大脑中动脉闭塞（MCAo）大鼠大脑后的命运。在体外，从培养基中去除生长因子和4-OHT后4周，c-mycER TAM 转基因转录降低了约75％。此外，免疫细胞化学和蛋白质印迹法显示c-MycER TAM 蛋白同时减少。为了检测转基因的体内转录，在MCAo损伤后4周植入了CTX0E03细胞（450,000），并分别通过qPCR和qRT-PCR分析了人类细胞的存活率和c-mycER 转录。 。结果结果表明，在所有移植的动物脑中均存在CTX0E03细胞，占注入的总细胞的6.3％至39.8％。植入前，CTX0E03细胞悬液每细胞含有215.7（SEM = 13.2）份c-mycER TAM 转录本。植入后，每个CTX0E03细胞的c-mycER TAM 转录本拷贝数在1周时已降至6.9（SEM = 3.4），在4周时已降至7.7（SEM = 2.5）。体内样品的亚硫酸氢盐基因组DNA测序证实，通过转基因启动子序列的甲基化，发生了c-mycER TAM 沉默。结论结论总之，结果证实了CTX0E03细胞在EGF，bFGF和4-OHT撤离后体外以及在MCAo大鼠脑中植入后均下调了c-mycER TAM 转基因表达。体内c-mycER TAM 转基因的沉默为潜在的临床应用提供了CTX0E03细胞的另一安全特性。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号