The fruit fly Drosophila serves as an important model organism for human biology. In addition to the genome sequence of D. melanogaster, the genome sequences of 11 other Drosophila species have been made publicly available over the past two years. These genome sequences have brought unprecedented opportunities to study functions of genes and their implications in development and disease. Gene functions are often inferred from specific perturbation of the genetic makeup of an organism and subsequent analysis of the effects on the organism. Two powerful methods have been established in the past for such analysis in Drosophila: permanent genetic transformation with transposable genetic elements and transient specific gene silencing through RNA interference (RNAi). Both methods require reliable and rapid injection of DNA and doublestranded RNA (dsRNA), respectively, at the earliest stages of embryonic development.
Within the frame of this NSF CAREER project, two automated, MEMS-based Drosophila embryo injection systems termed 'Search and Inject' and 'Feed and Inject' will be developed to enable high-throughput screens for gene functions in Drosophila embryos. 'Search and Inject' will support RNAi experiments; 'Feed and Inject' will support genetic transformation experiments. The automated injection technologies will lead to an approximately 20-fold increase in experimental throughput compared to state-of-the-art manual injection procedures. Development of robust technologies beyond proof of principle will enable their dissemination and widespread use within the Drosophila research community. The injection systems developed under the NSF CAREER award will be complemented with a system for high-throughput, confocal imaging of Drosophila embryos as well as image analysis software for reliable, automated recognition of phenotypes due to gene silencing.
Application of these new tools will lead to a better understanding of molecular mechanisms of development and disease in humans, with expected significant impact on new therapies and improvement of the state of public health. The proposed research will advance fundamental engineering knowledge regarding design, fabrication, packaging, and application of MEMS devices. The generated knowledge can help create systems for automated handling of DNA, RNA, other biochemical reagents, cells, oocytes, embryos, as well as micro- and nanoparticles, with widespread applications in biological research, biotechnology, drug discovery, high-throughput screening, and medical diagnostics.
Educational and outreach activities are designed to interest middle school, high school, and undergraduate students early on in a career in science and to educate a new breed of engineers who can creatively identify and address technological needs in biology and medicine. Outreach activities include a ten-week-long undergraduate summer research program for minority students, a six-week-long summer research program for high school students, and a weekend workshop for middle school students. Research is also directly integrated in two new, interdisciplinary classes: 'BioMEMS and Biomedical Nanotechnology' and 'Stem Cell Engineering'.
Advances in biology nowadays critically depend on new technologies. General goals of the activities under this grant were to develop micro- and nanotechnology-based devices to support biological research, to train engineering students to perform interdisciplinary research, and to develop new classes at the undergraduate and graduate level that prepare students for innovative research and product development at the interfaces of technology, biology and medicine. Research focused in particular on establishment of microelectromechanical system (MEMS)-based technologies for automated injection of embryos of the fruit fly Drosophila, one of the most important model organisms for human development and disease. The grant also partially supported pilot projects in the field of stem cell engineering, aimed at exploring neural stem cell biology and use of neural stem cells in preclinical, therapeutic approaches. Two new automated systems for injection of Drosophila embryos were created. In the first system, embryos are automatically retrieved from an off-chip reservoir, injected inside a microfluidic device with the help of an integrated, surface micromachined microinjector, and afterwards stored in a second off-chip reservoir. This system is particularly useful for end-on injection of Drosophila embryos and can support generation of genetically modified Drosophila lines where injected embryos are allowed to develop into adults. In a second injection system, embryos are attached to a glass slide. Machine vision is used to automatically screen the slide for embryos. Motorized stages are used to move slide and a MEMS injection chip relative to each other in order to inject embryos at their center points. This injection system is well suited for screening applications where injected embryos are imaged during their remaining embryonic development. In order to perform automated screens, the second injection system was complemented with an automated LEICA TCS SP5 confocal fluorescence microscope system that imports embryo position data from the injection system and automatically acquires time-lapse z-stacks of injected embryos after glass slide transfer. In order to automatically predict phenotypes, for example, due to gene silencing through RNA interference after injection of embryos with suitable double-stranded RNA, image analysis algorithms were developed and implemented. The developed systems fill urgent technological gaps and represent important steps toward automation of systematic experimentation with Drosophila embryos in particular and multicellular organisms in general. Experimentation with model organisms helps to better understand the molecular mechanisms of human development and human disease mechanisms. Stem cells, on the other hand, hold tremendous promise for use in regenerative medicine approaches, aimed at repairing or replacing tissues or organs that were damaged due to disease or injury. The grant supported development of polymeric microcapsules with cell-instructive properties, used for culture, expansion and lineage-specific differentiation, respectively, of adult neural stem cells. Microfluidic systems were created for automated encapsulation of stem cells as well as for assembly of heterogeneous, microcapsule-based tissue engineering constructs. Culture platforms based on micropatterned silicon substrates and spun nanofibers, respectively, were used to examine the effects of topographical cues on neural stem cell differentiation. Chitosan-coated, iron-oxide nanoparticles were synthesized that are efficiently internalized by neural stem cells through endocytosis and enable tracking of stem cells by magnetic resonance imaging (MRI). Also, neural stem cells were genetically modified, for example, to inducibly express the proneural transcription factors Olig1 or Ngn1, forcing neural stem cells to differentiate predominantly into oligodendrocytes or neurons. All these technologies can be applied to investigate what environmental factors stem cells respond to and how to design microenvironments so that stem cells are instructed to proliferate, differentiate or migrate in support of a specific therapeutic aim. The grant supported entirely or partially training of four doctoral students, one Master student and twelve undergraduate students in the PI’s lab. Dissemination efforts include publication of five journal articles, one book chapter and 24 conference presentations. As an outreach effort, the PI organized a summer undergraduate research program in bioimaging, engineering and technology, targeting especially minority students. The PI organized twice the one day research symposium ‘Bioimaging Day’ at Carnegie Mellon University, bringing together researchers who produce bioimage data in their research programs and researchers who specialize in image analysis. ‘Bioimaging Day’ also represents a unique educational opportunity for attending students. The PI served for three years as advisor to the newly founded undergraduate student chapter of the national BMES society at Carnegie Mellon. The chapter informs interested students about biomedical careers, forms a biomedical student community through many social and professional events, and reaches out to the general public. The PI developed the three new courses ‘Introduction to Biomedical Engineering’, ‘Biosensors and BioMEMS’ and ‘Stem Cell Engineering’ and educated during the award period more than 400 students. Students learned in all classes about the frontiers and fundamentals of interdisciplinary research at the interfaces of technology, biology and medicine. The PI’s research was discussed and showcased as integral part of each class.
