Model Systems: Innate Immunity

In the last year the Ozinsky group focused on five projects:

Measuring cytokine gene expression heterogeneity in murine macrophages
A procedure for single-cell mRNA analysis that combines flow cytometry, cDNA synthesis, and measurement by real-time PCR has been developed to address cell-to-cell heterogeneity in macrophage gene expression. With this analysis, the heterogeneity in cytokine gene expression in a population of murine bone marrow-derived macrophages has been evaluated. Surprisingly, there were no obvious coordinate expression response patterns for a set of pro-inflammatory genes. PMCID: PMC2711328 . A free article.

Purifying human breast cancer stem cells and identifying marker gene co-expression in single cells
The presences of stem cells are hypothesized to contribute to the malignancy and radiotherapy resistance of breast tumors. Since the behavior of cancer stem cells is thought to mimic many features of embryonic stem cells, the regulatory programs initiated by transcription factors such as Oct4, Nanog and Sox2 are being studied. These and other candidate genes from sets of individual human mammary epithelial (HMLE) cells are being measured, using a method developed to perform multiplexed gene expression measurements with single-cell resolution on macrophages.

Developing a microfluidic device to perform multiplexed ELISA measurements on single cell samples (with ISB, NIST, Caltech, and Tampere University of Technology) A scalable, high-density bead-based protein capture array platform is being developed that will permit the simultaneous measurement of multiple proteins from individual cells. The goal for the proposed automated microfluidic measurement platform is to have the capacity to routinely and reliably measure 16-20 proteins from 64-128 samples.

Purifying cells based on in situ phenotype using the Cyntellect LEAP instrument (with ISB and Seattle Biomedical Research Institute) Preliminary experiments are being performed using a laser-enabled analysis and processing (LEAP) instrument (Cyntellect) to assess the feasibility of purifying cells based on phenotype such as their proximity to other cells, and activity such as T cells mediated cytolysis.

Epitope tag technology applied to identifying host proteins suppressed by influenza (resulting in Cancer Research Institute/Irvington Fellowship award to postdoctoral fellow Shari Kaiser) An epitope tag for influenza virus was devised to be able to detect host proteins that are suppressed during influenza infection. Through other funding, these techniques are being applied to mass spectrometry to identify host proteins that differentially interact with a series of matched variants of influenza virus genes with different virulence potentials