Journal: Scientific Reports
Article Title: A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis
Figure Lengend Snippet: Transcriptional analysis of RNA recovered from wild type and allelic exchange mutant E . chaffeensis organisms assessed by RT-PCR. ( A ) RT-PCR products from wild type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb plus molecular weight DNA markers resolved; +, genomic DNA from wild type E . chaffeensis was used as the template; -, negative control reaction with no template added). ( B ) As in panel A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiments included genomic DNAs as the templates from W, M and R. (0.38 kb amplicons are expected for DNA templates in PCRs of W and R and 1.6 kb product is expected for M DNA as the template.) ( C ) Mutations to inactivate and restore the gene activity in Ech_0379 did not alter the gene expression from its neighboring genes. Semi-quantitative RT-PCR assays were performed at 30, 35 and 40 PCR cycles for Ech_0378, Ech_0379 and Ech_0380 for wild type, gene inactivation and gene rescue mutant organisms and the data for 35 cycles were presented. W, M and R had similar quantities of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R, while absent for M. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).
Article Snippet: RNA analysis by RT-PCR to verify the loss and restoration of transcription Total RNAs from wild type and mutant E . chaffeensis organisms grown in ISE6 or DH82 cell cultures were isolated by following the Tri-reagent total RNA isolation method (Sigma-Aldrich, St. Louis, MO).
Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Negative Control, Activity Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction