Genomics
Genomics techniques are used to study cellular gene expression and regulation. Two major genomics techniques are RNA
expression analysis and genome-wide location analysis.
RNA expression analysis measures the mRNA content of a cell through the amplification and labeling of RNA message in a biological sample. Labeled, fragmented, and amplified complementary RNA is hybridized to an oligonucleotide microarray and specific signal is measured through image analysis. An increase in hybridization signal indicates an increase in RNA message production during various cellular conditions.
Genome-wide location analysis techniques reveal the binding of transcription factors to genomic DNA, indicating control of gene expression by specific protein factors. We have been using two approaches for genome-wide location analysis, ChIP-on-chip and ChIP-Seq. With both techniques cells are fixed, the genomic DNA is sheared to ~500 bp in size, transcription factors are immunoprecipitated using specific antibodies, and isolated genomic DNA is extracted to remove proteins, etc. This immunoprecipitated DNA is then processed differently for each technique. For ChIP-on-chip the DNA is amplified, fragmented, labeled, and hybridized to a tiled genomic DNA oligonucleotide microarray. Specific binding of a transcription factor to a DNA sequence results in enrichment of this specific sequence in the immunoprecipitated DNA. Hybridization of this enriched sequence to an array results in increased signal intensity that is quantified through image analysis. For ChIP-seq the DNA is modified with adapters on both ends, size-selected, PCR-amplified, and then each fragment is sequenced using the Genome Analyzer system from Illumina. The fragments are aligned to the mouse genome and the number of fragments that align to particular sequences are tallied. An increased number of fragments aligning to a particular area in the genome represents specific binding of a regulatory protein to the DNA. The major difference between these two genome-wide location analysis techniques is that ChIP-on-chip measures the enrichment of the certain sequences that are tiled on the array while ChIP-seq measures, potentially, all the sequences that are in the sample without restriction to chosen sequences. Therefore ChIP-Seq has the advantage that it may uncover previously unknown regulatory sequences in the mouse genome.
Genomics and the Innate Immune Response
We are using genomics techniques to study the regulation of the innate immune response to whole pathogens and
pathogen-associated molecular pattern (PAMPs) that are known to activate Toll-like receptors. We are studying the
global transcriptional response of of bone marrow-derived macrophages (BMDMs) and dendritic cells (DCs) to challenge
with whole pathogens and individual PAMPs. We are also studying the global transcriptional responses of immune cells
isolated from mice that are challenged in vivo with intact pathogens or individual PAMPs. We are performing
ChIP-on-chip and ChIP-Seq with specific transcriptional regulators identified through the analysis of global
transcriptional responses in these model systems. These experimental approaches combined with computational analysis
will identify novel regulators and uncover network relationships among these regulators. The Genomics Core is also
using these techniques to analyze novel mutants generated by the Forward Genetics Core.