PUBLICATION

RNA Sequencing of FACS-Sorted Immune Cell Populations from Zebrafish Infection Models to Identify Cell Specific Responses to Intracellular Pathogens

Authors
Rougeot, J., Zakrzewska, A., Kanwal, Z., Jansen, H.J., Spaink, H.P., Meijer, A.H.
ID
ZDB-PUB-140831-4
Date
2014
Source
Methods in molecular biology (Clifton, N.J.)   1197: 261-74 (Journal)
Registered Authors
Kanwal, Zakia, Meijer, Annemarie H., Rougeot, Julien, Spaink, Herman P., Zakrzewska, Ania
Keywords
none
MeSH Terms
  • Animals
  • Animals, Genetically Modified
  • Disease Models, Animal*
  • Flow Cytometry*
  • Mycobacterium marinum/immunology*
  • Sequence Analysis, RNA*
  • Zebrafish/immunology*
  • Zebrafish/microbiology
PubMed
25172286 Full text @ Meth. Mol. Biol.
Abstract
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.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping