Arrays for Cloning Growth Suppressed Cells

描述（由申请人提供）：为研究特定基因对细胞生理学的影响而创建的分子工程细胞是生物医学发现的基本工具。当代癌症研究的一个主要焦点是通过创建表达肿瘤抑制基因或具有癌基因敲除的细胞系来研究细胞存活和复制的分子机制。虽然对于研究癌细胞特征的不受控制的复制至关重要，但由于其生长劣势，这些细胞系极难产生和维持。基于微加工细胞阵列的新技术有可能为这些细胞工具的日常创建和维护提供一个使能平台。在目前的拨款申请中，一个跨学科的团队将开发可释放的细胞“筏”阵列，以克服分离和维持生长不利的克隆集落的技术挑战。将使用易于实施和廉价的制造技术来制造阵列。将优化阵列设计和材料以用于转染和扩增阵列上的细胞。将进行关键生物学控制，以确认细胞健康、活力和克隆性。将基于标准显微成像开发用于阵列分析和克隆集落选择的方法。将优化一个简单的机械系统，以释放靶细胞所在的单个筏。这项工作将包括硬件和软件的设计和测试，以实现半自动分析和细胞分离。将建立和维持肿瘤抑制生物学研究所需的细胞系，并通过与传统方法的比较来验证该技术。本研究中开发的基本创新将降低阵列制造成本，并将产生灵活，可靠和简单易用的技术。这种新的细胞克隆方法所产生的能力将导致对控制癌细胞生长和存活的新见解。 公共卫生相关性：这项工作的目标是开发一种细胞阵列平台，即使细胞携带抑制细胞生长的基因，也能分离和生长细胞。这项技术是研究肿瘤抑制基因和其他调节细胞生长的基因所必需的。分离和培养携带这些基因的细胞的能力将增强我们对肿瘤抑制基因的分子基础在癌症中的重要性的理解。

DESCRIPTION (provided by applicant): Molecularly engineered cells created to study the impact of specific genes on cell physiology are fundamental tools for biomedical discovery. A major focus of contemporary cancer research is the study of the molecular mechanisms underlying cell survival and replication by creation of cell lines that express tumor suppressor genes or have oncogene knockouts. While critical for the study of the uncontrolled replication that characterizes cancer cells, these cell lines are extremely difficult to generate and maintain due to their growth disadvantage. New technologies based on microfabricated cell arrays have the potential to provide an enabling platform for routine creation and maintenance of these cellular tools. In the current grant application, an interdisciplinary team will develop arrays of releasable cell "rafts" to overcome the technical challenges in isolation and maintenance of clonal colonies with a growth disadvantage. Easily implemented and inexpensive fabrication techniques will be used to manufacture the arrays. Array design and materials will be optimized for transfection and expansion of cells on the array. Critical biological controls will be performed to confirm cell health, viability, and clonality. Methods for array analysis and selection of clonal colonies will be developed based on standard microscopic imaging. A simple mechanical system will be optimized in order to release individual rafts on which target cells reside. This work will include the design and testing of hardware and software to enable semi-automated analysis and cell isolation. Cell lines needed for studies of tumor suppressor biology will be created and maintained, and the technology will be validated by comparison with conventional approaches. The fundamental innovations developed in this research will decrease the costs of array manufacture and will produce a flexible, reliable and simple to use technology. The capabilities engendered by this new method for cell cloning will lead to new insights into the control of cancer cell growth and survival. PUBLIC HEALTH RELEVANCE: The goal of the proposed work is to develop a cell array platform that enables cells to be isolated and grown even when the cells carry a gene that inhibits cell growth. This technology is needed for the study of tumor suppressor and other genes that regulate cell growth. The ability to isolate and grow cells carrying such genes will enhance our understanding of the importance of the molecular basis of tumor suppressor genes in cancer.