 |
|
|
|
|
|
Research and Scientists
|
|
|
|
|
|
|
|
|
|
|
|
Model Systems: Innate Immunity
Adrian Ozinsky
is seeking to define the structure of the Toll-Like Receptor (TLR) signaling pathway and gene regulatory network.
Significance:
We are developing experimental assays and measurement platforms to allow us to evaluate immune cell functional
responsiveness to pathogenic stimuli at the level of individual cells. This capability will provide novel
insights into the range of differential responses cells defined as being in the same cellular 'class' (e.g.
macrophages, T-cells, etc), as well as the ability to directly interrogate gene regulatory and cell signaling
mechanisms in rare cell types such as memory T-cells.
Research and Results:
Single Cell PCR
Few quantitative biological measurements are made with single-cell or sub-cellular
resolution. Rather, most standard biological assays assess the integrated response
of thousands to millions of cells, thereby averaging the biological activity of a
cellular population. For example, microarray measurements of innate immune cells such as
macrophages stimulated by pathogens demonstrate that the expression of approximately 2000
genes is altered. The degree to which the responses of individual cells differ from
the population average remains unclear.
In order to disentangle the cell-cell heterogeneity confounding assessment of innate
immune responses to microbes and microbial products, we have developed a new and
straightforward protocol for single-cell mRNA analysis that combines the power of high-speed
cell sorting (ability to isolate rare cells based on phenotypic markers), robust cDNA synthesis,
and quantitative mRNA expression measurements by real-time PCR. The power of this procedure
goes well beyond what is currently available for assessing immune cell responses by
immunohistochemistry, flow cytometry or microarray analysis. The procedure permits the
assessment of many more markers (~50) than is routinely measured by flow cytometry
(<5), without being limited by the availability of antibody reagents. The current scale
of this procedure is the measurement of a panel of ~25 innate and adaptive immune genes in
each of 200-400 samples, in a microtiter plate format with an experimental turnaround/cycle
time of <2 days. This approach provides for facile detection of highly expressed genes at the
single-cell level (such as cytokines responding macrophages) and a sensitivity of detection
of as little as 20-30 mRNA copies/cell.
We are also implementing a semi-automated pipeline for cDNA synthesis and mRNA expression
measurements by real-time PCR. In addition to increasing the capacity to process the required samples,
automation will standardize the procedure, minimize errors, and reduce the reagent volume
(and thus cost) of each assay.
Development of Microfluidic Technology for Single Cell Analysis of Leukocytes
In collaboration with Carl Hansen,
we are developing microfluidic devices capable of long-term mammalian cell culture at the nanoliter scale, with
the ultimate goal of culturing single cells "in-chip" for subsequent biological manipulation and analysis.
Long-term time-lapse imaging studies of leukocytes within prototype devices are being routinely performed
with the Imaging Core resources (add link to Imaging and Microfluidics Core), along with functional
assays to assess whether proper cellular responses are retained following stimulation within these
small volumes. In parallel, we are developing microfluidic assays to perform multiplexed protein
measurements within nanoliter volumes that can quantitatively measure secreted cytokines and chemokines
profiles secreted from single leukocytes in response to whole pathogens or pathogen-associated molecular
patterns. By monitoring the kinetic profiles of multiple secreted proteins in parallel over time, we can
gain insight into the heterogeneity of immune regulatory cells such as macrophages, and how different
responses at the single-cell level may mediate cell-specific effector functions that would be otherwise
masked by population-based cell assays.
Central Dogma-Tagging (CD-Tagging)
To better understand the diversity of macrophage responses to microbes, it will be essential to
incorporate imaging modalities to quantify morphological changes or altered patterns of protein
expression (visualized by fluorescent reporters such as GFP) that can reveal cell-to-cell heterogeneity
within cell populations. We have begun studies to address how to scale up fluorescent imaging experimentation
for high-throughput analysis of cellular phenotype. A comprehensive screen based on CD-tagging of
macrophages is being undertaken to identify novel transcriptional regulators of the innate and adaptive
immune response. This method uses a viral vector to randomly introduce a GFP coding construct flanked
by exon consensus splice sites into the genome of the target cells at a frequency such that most cells
contain no more than one insert. Successful insertions result in a GFP fusion protein under the control
of the endogenous promoter. A library of GFP-positive cells is generated by single cell sorting, and
distributed into optical quality microtiter plates. We are creating a CD-tagged macrophage library, and
we will screening this library for labeled proteins whose cellular localization changes under a
variety of immune stimuli.
|
|
|
|
|
|
|
|
|
|
|
|
|
Institute for Systems Biology
CSB Home
Center for Systems Biology at the Institute for Systems Biology
