Pbluescript 2 sk

Digoxigenin (DIG)-labeled antisense RNA probes were synthesized using DIG RNA labeling mix (Roche) and T3 (Fermentas), T7 (Fermentas) or SP6 (Roche) RNA polymerase according to the manufacturer's instructions. For the kni probe, an EST clone GH19318 [66] (link) was used as a templates. For the vvl probe, a portion of vvl gene was amplified by PCR with cDNA derived from w¹¹¹⁸ larvae and the following primers: vvl_PA_CDS_F (5′-ATGGCCGCGACCTCGTACATGAC-3′) and vvl_PA_CDS_R (5′-CTAGTGGGCCGCCAACTGATGC-3′). For the mld probe, a portion of mld gene was amplified by PCR with the plasmid mld-pUAST [21] (link); a gift from S. M. Cohen and the following primers: mld_CDS_1_F (5′-ATGAGTGCCAACCGAAGAAGACG-3′) and mld_CDS_1_R (5′-CATCTGAGATTGGTCATGAGATTGTACTTGAGG-3′). PCR products containing the vvl and mld fragments were subcloned into SmaI-digested pBluescript II SK(-) and pCRII-Blunt-TOPO (Invitrogen), respectively, and then used as the templates for synthesizing RNA probes. Fixation, hybridization and detection were performed as previously described [8] (link), [67] (link).

Escherichia coli DH5α and HB101 strains were used for cloning procedures in pBluescript II SK (+) (Invitrogen) vector or pJV53-zeo plasmid^{81 (link)} propagation were cultured in Luria Bertani (LB) liquid or solid media. When required kanamycin (Sigma), zeocin (Invitrogen), hygromycin (Invitrogen) and ampicillin (Sigma) were added to a final concentration of 50 μg/ml (kanamycin), 25 μg/ml (zeocin), 100 μg/ml (hygromycin and ampicillin). Mtb reference strain H37Rv and Mtb clinical isolates were grown in Middlebrook 7H9 liquid medium, supplemented with 0.2% glycerol (w/v) and 10% enrichments containing BSA (Sigma) 5% (w/v), glucose (Sigma) 2% (w/v) and NaCl (Sigma) 0.85% (w/v). For growth on solid medium, Middlebrook 7H11 agar medium was used supplemented with glycerol 0.5%, and 10% oleic acid-albumin-dextrose-catalase enrichments (OADC). When needed, kanamycin, hygromycin or zeocin were added to a final concentration of 20 μg/ml, 100 μg/ml, 25 μg/ml, respectively. Plates used for CFU counting of lung homogenates contained a PANTA (Becton Dickinson) antibiotic mixture. Inspection of Middlebrook 7H11 agar plates for CFU counting of different bacterial strains in various experimental conditions was undertaken after 3 and 5 weeks of incubation.