RNA Sequencing of FACS-Sorted Immune Cell Populations from Zebrafish Infection Models to Identify Cell Specific Responses to Intracellular Pathogens
The zebrafish (Danio rerio) is increasingly used as a model for studying infectious diseases. This nonmammalian vertebrate host, which is transparent at the early life stages, is especially attractive for live imaging of interactions between pathogens and host cells. A number of useful fluorescent reporter lines have recently been developed and significant advances in RNA sequencing technology have been made, which now make it possible to apply the zebrafish model for investigating changes in transcriptional activity of specific immune cell types during the course of an infection process.
Here we describe how to sequence RNA extracted from fluorescently labeled macrophages obtained by cell-sorting of 5-day-old zebrafish larvae of the transgenic Tg(mpeg1:Gal4-VP16);Tg(UAS-E1b:Kaede) line. This technique showed reproducible results and allowed to detect specific expression of macrophage markers in the mpeg1 positive cell population, whereas no markers specific for neutrophils or lymphoid cells were detected. This protocol has been also successfully extended to other immune cell types as well as cells infected by Mycobacterium marinum.
Key wordsRNA sequencing FACS Immune cells Zebrafish larvae dissociation Transcriptome analysis
This work was supported by the Marie Curie Initial Training Network FishForPharma (PITN-GA-2011-289209) and the project ZF-HEALTH (HEALTH-F4-2010-242048) funded by European Commission 7th Framework Programme, and by the SmartMix programme of the Netherlands Ministry of Economic Affairs and the Ministry of Education, Culture, and Science. Additionally, Z.K. was supported by the Higher Education Commission of Pakistan, and A.Z. was supported by a Horizon grant of the Netherlands Genomics Initiative.
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