CORE--Cell & Molecular Biology

细胞和分子生物学（CMB）核心的总体功能将是为该计划提供 每个项目都需要精心控制和表征的人类细胞培养物。服务 由核心提供的方法包括分离、培养、储存和分布初级黑素细胞， 角质形成细胞和成纤维细胞。此外，CMB Core还将培养和分发所有的黑色素瘤 细胞系也将表征B-RAF、N-RAS和PTEN中的DNA突变。CMB核心将 确保程序中使用的所有细胞不受支原体或其他污染物的污染 在有需要时进行定期测试及消毒。中巴核心会产生病毒， 用致黑素基因或shRNA转导原代黑素细胞以敲低DNA损伤 反应基因转导的细胞将表征这些基因的表达和功能， 敲减载体，其使用信息测定如蛋白质印迹分析。CMB核心将在 体外人皮肤重建物（也称为器官型皮肤培养物或皮肤等同物），其中 人造人皮肤在体外用初级黑素细胞的混合物在三维系统中产生， 角质形成细胞和成纤维细胞。这些皮肤重建将提供给项目1，用于体外分析， DNA损伤反应和项目3，用于在SCID小鼠背部植入，用于体内DNA分析 损伤响应CMB核心的PI（Shields博士）最近完成了一项由NCI赞助的为期两周的 在Wistar研究所与Meenhard Herlyn博士（国际公认的领导者， 黑色素瘤研究和项目外部顾问），以获得人类皮肤制备方面的实践培训 重建。细胞和分子生物学核心通过提供重要的信息服务于计划项目。 生物试剂来进行研究

The overall function of the Cell and Molecular Biology (CMB) Core will be to provide the program with carefully controlled and characterized human cell cultures that are needed in each project. Services provided by the core wjll include the isolation, culture, storage and distribution of primary melanocytes, keratinocytes and fibroblasts. In addition, the CMB Core will also culture and distribute all of the melanoma cell lines that will also be characterized for DNA mutations in B-RAF, N-RAS and PTEN. The CMB Core will ensure that all cells used in the program are free of contamination with mycoplasma or other contaminants by providing regular testing and decontamination when necessary.The CMB core will generate virus for transduction of primary melanocytes with melanomagenic genes or shRNA's to knockdown DNAdamage response genes. Transduced cells will be characterized for expression and function of these genes and knockdown vectors using informative assays such as western blot analysis. The CMB Core will generate in vitro human skin reconstructs (also referred to as orgariotypic skin cultures or skin equivalents) in which artificial human skin is created in vitro in a three-dimensional system with a mixture of primary melanocytes, keratinocytes and fibroblasts. These skin reconstructs will be supplied to Project 1 for in vitro analysis of DNA damage response and Project 3 for engraftment on the backs of SCID mice for in vivo analysis of DNA damage response. The PI of the CMB Core (Dr. Shields) recently completed an NCI-sponsored two-week training program at the Wistar Institute with Dr. Meenhard Herlyn (an internationally recognized leader in melanoma research and Program externaladvisor) to gain hands-on training in preparation of human skin reconstructs. The Cell and Molecular Biology core serves the Program Project by providing important biological reagents to program investigators.