Wednesday, September 18, 2024
9/18/2024
DESCRIPTION (provided by applicant) The sequencing of many genomes has led to the development of new technologies for studying gene expression in prokaryotic and eukaryotic cells. These techniques, especially gene expression profiling with DNA arrays are revolutionizing studies of microbial physiology, eg, cellular responses to different growth conditions and environmental stimuli, etc. These new methods can also be used to study bacterial and/or host gene expression during infections, as they facilitate transcriptional analyses of the interaction between bacterial pathogens and their host. The purpose of this grant application is to do an integrated study of bacterial and host gene expression during M. tuberculosis infection of macrophages and animals, using standard DNA array technology and by developing new methods, as well. The aims of the project are as follows: 1) Expression profiling of M. tuberculosis genes in vitro and during infection.We will use DNA arrays containing the entire M. tuberculosis4000 gene set (Mtb gene array) to study the levels of all the mRNAs of wild type and mutant M. tuberculosis. in vitro in response to various environmental stresses. The arrays will also be used to study the levels of mRNAs of wild type and mutant M. tuberculosis during infection of human and mouse macrophage cell lines. We will also develop molecular beacon derived methods described in specific aim 3 to study the expression of M. tuberculosis genes in different tissues during the infection of animals. 2) the second major aim is to do expression profiling of host genes during M. tuberculosis infections. For these studies, we will use Affymetrix or custom arrays of the human and mouse genomes to study host gene expression during M. tuberculosis infection of macrophages and during later stages of infecrtion in different tissues of infected animals. 3) the final aim is to develop new methods for studying M. tuberculosis gene expression during animal infections. These methods will use molecular beacons and related techniques for fixed arrays that will allow the detection and quantitation of multiple M. tuberculosis mRNAs during infection and will employ specific amplification during hybridization so that low levels of bacterial mRNAs can be specifically detected in the presence of high levels of host RNA.
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