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| Status |
Public on Jun 01, 2014 |
| Title |
Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis (RNA-seq) |
| Organism |
Danio rerio |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
MyD88 is an adaptor protein in Toll-like receptor and interleukin 1 receptor mediated signaling pathways that plays an essential role in activation of immune responses following pathogen recognition. We investigate that role in the zebrafish embryo model by using a zebrafish mutant line that contains a premature stop condon in the gene encoding MyD88, leading to a truncated protein that lacks domains important for its normal function. We infected these MyD88 mutants and wildtype individuals with Mycobacterium marinum to compare the resulting immune response by transcriptome profiling on total RNA isolated from single embryos. Autophagy regulator dram1 was identified as one of the MyD88-dependent genes.
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| Overall design |
This RNAseq analysis was used to determine the effect of a truncation of the MyD88 protein on the innate immune response of zebrafish embryos during infection with Mycobacterium marinum. Myd88 mutant and wild type embryos were derived by incrossing homozygous myd88 mutant parents (allele hu3568, van der Vaart et al., 2013, Disease models & mechanisms 6, 841-854) or their wildtype siblings. RNA was isolated from pools of 20 embryos at 4 days post infection (4 dpi). The following treatment groups were used: homozygous mutants mock-injected with PBS/2%PVP 4 dpi, (2) wildtype siblings mock-injected with PBS/2%PVP 4dpi, (3) M. marinum-infected homozygous mutants 4dpi, (4) M. marinum-infected wildtype siblings 4dpi. Embryos were grown at 28.5–30°C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200 colony forming units (CFU) of Mycobacterium marinum Mma20 bacteria into the caudal vein, or were mock-injected with buffer (PBS/2%PVP) as a control. After injections embryos were transferred into fresh egg water and incubated for 4 days at 28°C. After the incubation period, single embryos were snap-frozen in liquid nitrogen and RNA was isolated for RNAseq analysis.
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| Contributor(s) |
van der Vaart M, Spaink HP, Meijer AH |
| Citation(s) |
24922577, 25247677 |
| Submission date |
Jul 24, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Annemarie H. Meijer |
| E-mail(s) |
a.h.meijer@biology.leidenuniv.nl
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| Organization name |
Institute of Biology, Leiden University
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| Department |
Animal Sciences and Health
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| Street address |
Einsteinweg 55
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| City |
Leiden |
| ZIP/Postal code |
2333 CC |
| Country |
Netherlands |
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| Platforms (1) |
| GPL14875 |
Illumina HiSeq 2000 (Danio rerio) |
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| Samples (4)
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| This SubSeries is part of SuperSeries: |
| GSE49188 |
Autophagy regulator DRAM1 functions downstream of MYD88 in defense against tuberculosis |
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| Relations |
| BioProject |
PRJNA213244 |
| SRA |
SRP028229 |
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