The primary objective of this research project is to identify genes that are differentially expressed in vivo by virulent strains of M. tuberculosis using reverse transcriptions and PCR technologies. The use of RT-PCR to monitor differential gene expression requires rapid MTB isolation from infected mice followed by RNA extraction from the purified bacteria. AntiMTB monoclonal antibodies coupled with magnetic beads have been used successfully to separate MTB from solution contaminants and will be applied to tissue homogenates for bacterial isolations. High quality RNA has been isolated from MTB and preliminary evidence suggests it can serve as an adequate substrate for reverse transcription and PCR. Combination of these techniques should permit identification of differentially expressed genes by comparison of RNA products from culture grown MTB with those RNAs from MTB grown in the mouse.
