Gene expression in human cells is the process that translates the information embedded in agene into the synthesis of a cellular product. It is a critical aspect of both normal and pathological development of cells and tissues. Current bulk gene expression assays rely on molecules extracted from multiple cells or tissue samples, therefore containing various degrees of cellular heterogeneity. As a result, it is difficult to determine the regulatory relationship of genes in different phases of development from these 'cell population averaging' measurements. The investigators have previously demonstrated that a microfluidic technology can be used for extracting total messenger Ribonucleic acid (mRNA) from single-cells and synthesizing complementary Deoxyribonucleic acid (cDNA) on the same chip for high efficiency single-cell gene expression profiling and reduces the minimum detectable number of mRNA molecules. However, one of the current challenges of them microfluidic device is the difficulty of using pneumatic pumping and valving mechanism for single cell addressing, which is a time-consuming and labor intensive task. In this project a microfluidic platform will be developed for massively parallel single cell mRNA analysis for gene profiling applications. The proposed device integrates three functional regions on a single Polydimethylsiloxane (PDMS) microfluidic chip: a high speed microscale fluorescence activated cell sorter (µFACS), optoelectronic tweezers (OET) for massively parallel single cell manipulation, and 1000 microfluidic wells for single cell mRNA extractions and cDNA conversion. This device will solve the technical issues in integrating OET with microfluidic devices to enable massively parallel single cell manipulation. One thousand cells will be individually trapped and transported into microfluidic wells where cells are split, or lysed, for gene profiling analysis. Integrating OET allows eliminating the multiplexed microfluidic control network that has been proven extremely inefficient in single cell manipulation and replacing it with dynamic optical images that can be reconfigured in real-time. Success in this proposal will realize a low-cost, fully integrated microfluidic chip capable of conducting massively parallel gene profiling on 1000 single cells. Knowledge developed during the course of this project will be incorporated into the investigators? teaching activities at both the undergraduate and graduate levels. Results of this proposal will be published in international conferences and peer-reviewed journals and information will also be available on the investigator website. Minority graduate and undergraduate students will participate in these projects through independent research courses. Students involved in this project will be exposed to an excellent multidisciplinary training environment between the USC Medical School and UCLA Engineering School. The PI will also be involved with the outstanding outreach program (CEED) in UCLA to recruit underrepresented college students for constructing a 'Virtual Chemical Lab' allowing everyone in the world to control cells in investigators' lab through Internet.
72 Normal 0 false false false EN-US X-NONE X-NONE We have achieved our aims and goals of this project to develop a device for molecular profiling of a single mamalian cell. 1) We have applied knowledge extracted from single-cell molecular profiles to direct cell differentiation. We published a cover paper in PNAS (processding of the National Academy of Sciences) to describe how we can use molecular information extrcated from a single-cell to direct the fate of a mamalian cell. This paper demonstrated the utility and underscored the importance to perfrom molecular profiling on a single cell rather than extracting the averaging moleuclar chrcateristics of a population of cells. Limited by the methods and analysis devices, current molecular biology studies often only study the molecular characteristics of a population of cells. Normal 0 false false false EN-US X-NONE X-NONE 2) We have demonstrated that the molecular characteristics obtained from a single-cell with our platform have similar quality as these from standard cell lysate, a mixture of millions of cells. With a publication in PLoS One, we demosntrtaed that our devices can measure gene expression levels within a single-cell in similar quality as these mesaured in cell lysate, a mixture of millions of cells. 3) With this NSF award, we also have supported a female student to participate in our daily research activities. The PI plans to expand the educational research activities by including more high school students for summary research and undergraduate students for biological research with future NSF supports.
