Microarrays and Their Use To Study Mammalian Development

Project: Research project

Project Details

Description

DESCRIPTION (Applicant's Abstract): The goals of the experiments outlined in
this proposal are to measure global temporal and spatial patterns of gene
expression during commitment and terminal differentiation. It has been made
clear from numerous studies that cells committed to different lineages differ
in the pattern of genes they express. A fundamental question concerning
developmental biologists is to dissect just how different growth factors and
signaling systems within stem cell populations' act to generate differentiated
phenotypes. It is exceedingly clear that complex problems like embryonic
development are performed by groups of molecules that function in concert. If
we are to understand what is happening during commitment and lineage
specification, we need to look at how all the players interact with one
another. To do this we need methods to simultaneously study thousands of genes
or gene products. I have set up a functional genomics program here at AECOM
that consists of a working cDNA microarray facility that currently uses glass
slides containing 9000 different genes for quantitative measurements of global
patterns of gene expression. I propose to use this system as a tool to study
mammalian cell culture systems that serve as models for differentiation into
neural, glial and cardiac myocyte cells. The strategy we will employ is to
first measure gene expression patterns in cells during the time course of
differentiation. In subsequent experiments we will genetically modify the cells
to create inducible systems to individually measure the portion of the entire
program that a specific transcription factor or signaling system contributes to
the overall developmental program. We propose to create and study changes in
gene expression in P19 lines and ES cells that have been stably transfected
with inducible alleles of known neurogenic and cardiogenic transcription
factors to dissect transcriptional hierarchies leading to terminal
differentiation. In this way, we hope to understand how each component of a
developmental program interacts, synergizes with, and regulates the activity of
other components. Bioinformatics and gene clustering tools will be critical to
integrate these results in the framework of normal differentiation. We have
used the embryonal carcinoma (EC) F9 cell line differentiating into parietal
endoderm in response to retinoic acid (RA) plus dibutyrl cyclic AMP as our
initial model system because it has been extensively studied in the past and we
can compare microarray results with those obtained by more traditional methods.
In addition, we recently completed a study of 16 time points from 1 hr. to 13
days of RA induced neural and glial cell differentiation in P19 cells and 10
time points from l to 10 days following DMSO induced cardiocyte differentiation
in P19clone6 cells. The results of these experiments and those with F9 cells
outlined in the preliminary results section of this proposal validate the
experimental approach mentioned above. The approach we are using should
implicate functional roles for a huge number of new genes for us and others to
study in greater detail.
StatusFinished
Effective start/end date6/1/015/31/02

Funding

  • National Institute of Neurological Disorders and Stroke: $410,289.00

ASJC

  • Genetics
  • Developmental Neuroscience
  • Molecular Biology
  • Cell Biology
  • Developmental Biology

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