Characterization of the zebrafish embryonic host response to systemic bacterial infection with Salmonella typhimurium wild type strain (SL1027) and its isogenic LPS O-antigen mutant Ra (SF1592) by means of a time-resolved global expression analysis.
Overall design
All infection experiments were performed using mixed egg clutches from three tanks of AB strain zebrafish. Embryos were staged at 27 hours post fertilization (hpf) by morphological criteria (Kimmel et al., 1995) and approximately 250 cfu of DsRed expressing S. typhimurium wt and Ra mutant bacteria were injected into the caudal vein close to the urogenital opening. As a control an equal volume of PBS was likewise injected. Injections were controlled using a Leica MZ Fluo 3 stereomicroscope with epifluorescence attachment together with a Femtojet microinjector (Eppendorf) and a micromanipulator with pulled microcapillary pipettes. Pools of 20-40 embryos were collected at 2, 5, 8 and 24 hours post infection (hpi). For the microarray analysis, the whole infection procedure was preformed in triplicate on separate days. The triplicates are marked A,B and C. The order of injecting wt bacteria, Ra bacteria and PBS control was randomized in the different experiments. The general reference sample is a mixture of all RNA samples from this infection study and it is named Common Reference (CR).
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