Transposon site hybridization (TraSH) screen in the lungs

We wanted to identify Francisella tularensis bacterial mutants that are negatively selected in vivo in the lungs of mice. Mice were infected with a Francisella transposon mutant library where each gene in the genome has been mutated via the insertion of a kanamycin resistance cassette with 2 outward facing T7 promoters. 2 days post infection, infected lungs were harvested and the bacteria present in the infected lungs were collected. Bacterial genomic DNA was isolated and subjected to an in vitro T7 transcription reaction, reverse transcribed and the resulting cDNA was hybridized to our Francisella microarray. Infection: The goal of the study was to identify Francisella genes that are negatively selected in the lungs of mice post infection with a Francisella transposon mutant library. Resulting bacterial cDNA was hybridized to the Francisella microarray. disease_state_design