Investigation Title Transcription profiling of mouse lung from C57BL6 and DBA2 parental strains and D2.B6-Chr7 and D2.B6-Chr19 congenic mice following aerosol infection with Mycobacterium tuberculosis for 30 and 70 days Comment[Submitted Name] Transcriptional profiling in the lungs of C57BL6 and DBA2 parental strains and D2.B6-Chr7 and D2.B6-Chr19 congenic mice following aerosol infection with Mycobacterium tuberculosis for 30 and 70 days Experimental Design time_series_design disease_state_design in_vivo_design strain_or_line_design co-expression_design transcription profiling by array Experimental Design Term Source REF The MGED Ontology The MGED Ontology The MGED Ontology The MGED Ontology EFO Comment[AEMIAMESCORE] 4 Comment[SecondaryAccession] Comment[ArrayExpressReleaseDate] 2009-02-20 Comment[ArrayExpressAccession] E-MEXP-1942 Comment[MAGETAB TimeStamp_Version] 2010-08-10 11:21:45 Last Changed Rev: 13058 Experimental Factor Name strain infect time Experimental Factor Type strain infect time Experimental Factor Term Source REF Person Last Name Marquis Person First Name Jean-François Person Mid Initials Person Email jf_marquis@hotmail.com Person Phone 514-398-2489 Person Fax 514-398-2603 Person Address Biochemistry, Bellini Building, 3649 Promenade Sir William Osler, Room 3830, Montréal, Québec, H3G 0B1, Canada Person Affiliation McGill University Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2009-02-20 PubMed ID 19265154 Publication DOI 19265154 Publication Author List Marquis, Jean-Francois; LaCourse, Ronald; Ryan, Lynn; North, Robert J.; Gros, Philippe Publication Title Genetic and Functional Characterization of the Mouse Trl3 Locus in Defense against Tuberculosis Publication Status submitted Publication Status Term Source REF The MGED Ontology Experiment Description To gain insight into the host cell types, cellular and molecular pathways possibly involved in the differential permissiveness to pulmonary replication of M. tuberculosis, we carried out transcript profiling studies on M. tuberculosis-infected lungs from congenic and parental strains. We were particularly interested in two groups of transcripts. The first group consists of transcripts which expression in the lung is regulated in response to M. tuberculosis infection (global response to infection), and that is obtained by comparing transcripts profiles of infected vs. uninfected lungs. The second group of transcripts is associated with increased resistance to M. tuberculosis infection of B6 and D2.B6-Chr7 mice. That list consists in the overlap between the lists commonly expressed in response to infection between resistant B6 and D2.B6-Chr7 but that show a significant difference in modulation when compared to infected susceptible D2.

In these experiments, B6, D2 as well as the D2.B6-Chr19, and D2.B6-Chr7 congenic lines were infected with M. tuberculosis and lungs were harvested at day 30 and day 70, and RNA was prepared. Three independent RNA samples from each group were converted to labeled cDNAs and hybridized to Affymetrix oligonucleotides arrays (Mouse Genome 430 2.0 array). Hybridization results were analyzed with the Genesifter analysis program to characterize changes in gene expression. Protocol Name P-MTAB-2567 P-MTAB-2568 P-AFFY-2 Affymetrix:Protocol:Hybridization-Unknown P-AFFY-6 P-MTAB-2566 Protocol Type nucleic_acid_extraction grow labeling hybridization feature_extraction bioassay_data_transformation Protocol Description Total RNA was extracted according to the manufacturer's recommendations (see Trizol protocol).
(Parameters: Extracted product = total_RNA, Amplification = none) Animals: Inbred, pathogen free, C57BL/6J (B6) and DBA/2J (D2) male mice were purchased from the Trudeau Institute Animal Breeding Facility. Mice were free of common viral pathogens as determined by routine testing performed by the Research Animal Diagnostic and Investigative Laboratory, University of Missouri (Columbia, MO, USA). Trl3 (Chromosome 7; Chr.7) and Trl4 (Chromosome 19; Chr.19) congenic mouse strains were generated using a speed-congenic (marker-assisted) approach. In this protocol, successive F1 backcross males were genotyped to identify individuals with the least residual donor genomic DNA, and were selected for further backcrossing. In these mice, the Chr.7 (Trl3) or Chr.19 (Trl4) segments from B6 strain was introgressed onto the genetic background of the D2 strain. For Trl3, the BXD19 recombinant inbred strain was used as the initial donor for the proximal portion of Chr.7 (D7Mit178-D7Mit193), while for Trl4 the BXD9 strain was the initial donor of the distal portion of Chr.19 (D19Mit69-D19Mit137). Once at the N4 generation, heterozygotes were intercrossed to generate the homozygote congenic lines and also to produce the double congenic line. Genotyping was carried out using tail genomic DNA and sequence-specific oligonucleotide primers. All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of the Trudeau Institute.



Infection with M. tuberculosis: M. tuberculosis strain H37Rv was obtained from the Trudeau Mycobacterial Culture Collection as a frozen (-70°C) log phase stock dispersed in Proskauer and Beck medium (Difco) containing 0.01% Tween 80. For each experiment, a vial was thawed, subjected to 5-s ultrasound to break up aggregates, and diluted appropriately in PBS containing 0.01% Tween 80. Mice, 8 to 10 weeks of age, were inoculated with 100 colony-forming units (CFUs) by the aerosol route in a Middlebrook airborne infection apparatus (Tri Instruments, Jamaica, NY).



mRNA expression studies: Mice were sacrificed (by cervical dislocation), and their lungs were rapidly homogenized in TRI REAGENT (SIGMA) using a polytron. Total RNA was extracted according to the manufacturer's recommendations.




(Parameters: time unit = seconds, temperature unit = C) Title: Affymetrix in vitro transcription. Description: Title: Affymetrix Generic Hybridization. Description: Title: Affymetrix CEL analysis. Description: Standard RMA normalization Protocol Parameters Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF mo The MGED Ontology SDRF File E-MEXP-1942.sdrf.txt Term Source Name NCI_thesaurus EFO The MGED Ontology NCBI Taxonomy ArrayExpress The MGED Ontology EFO mo Term Source File ncithesaurus.obo.alt http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ncbi.nlm.nih.gov/Taxonomy/ http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version Comment[AEExperimentType] transcription profiling by array