This project requests funding to upgrade equipment and develop high-throughput analysis algorithms to clone and analyze CRISPR/Cas9 transgenic recombinant cells. These cells are required to address the Specific Aims in NIH grant R01GM069429.
We have been successful in generating preliminary CRISPR/Cas9 cells that express kinesins and regulators of microtubule dynamics at endogenous levels as part of project R01GM069429. We have found that functional analysis of proteins in these cells is much more physiologically relevant than the more commonly used transfection and expression methods. This is in part because the proteins are at the appropriate level in the cell and in part because there is no contaminating activity from endogenous protein because the modifications for visualization and experimental manipulation are performed on the endogenous gene. One disadvantage is that the physiologically relevant levels of protein is challenging to image in cells. We have determined that with some minor modifications to our existing instruments we can significantly improve the production and analysis of these cell lines. Furthermore, we can develop high-throughput algorithms to analyze the cells due to their uniformity as a clonal cell line.
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