The production of murine models by means of gene-targeting embryonic stem cells is a fundamental tool in biomedical discovery. The selective targeting of a specific gene for removal or replacement through homologous recombination is widespread and has led to many new insights in biology and medicine. Furthermore, this advance in genetically modifying organisms is expected to lead to improved models of human disease and the creation of whole-animal systems for pharmacologic screening. An interdisciplinary group of investigators has been assembled to develop a single platform to generate the gene-targeted cells needed to produce genetically modified animals. This platform will integrate state-of-the-art microtechnologies consisting of microfabricated cell sorting arrays, miniaturized high-throughput DNA analysis techniques, and microfluidic-based DNA separations. Construction and optimization of the individual components of the proposed platform will be accomplished in four aims. First we will focus on the development and characterization of cell sorting arrays. Critical biological controls to validate each step in the development of this technology will be performed. Second we will optimize the sampling and high-throughput analysis by polymerase chain reaction (PCR) to screen potentially gene-targeted cells. Third, we will develop an integrated microfluidic system to assess screened cells for homologous recombination of the target gene. This device will perform DNA extraction/purification, restriction enzyme digestion, probe hybridization, and hybridized DNA separation. Finally, each component technology will be utilized to produce a genetically modified mouse in parallel with conventional technologies, and the two technologies will be compared.
This research develops new technologies to generate the gene-targeted cells in decreased time, at lower costs, and with a higher success rate than conventional technologies. These gene targeted cells are needed to produce genetically modified animals which are expected to lead to improved models of human disease and to create whole-animal systems for pharmacologic screening.
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