Project

Ongoing inflammatory response of zebrafish in vivo: Role of Soluble and Transmembrane TNFα (36 ECTS)

Background

Tumor Necrosis Fαctor-alpha (TNFɑ) is a pleiotropic cytokine involved in several aspects of the immune system and in particular the orchestration of inflammatory responses. TNFa is expressed in a soluble (s) or transmembrane (tm) form. While sTNFα is generally associated with inflammation, the role of tmTNFɑ is not well understood. We aim to determine the role of sTNFα and tmTNFɑ during inflammation, with special focus on macrophage activation.

Objectives

We are interested to study the ongoing inflammatory response of zebrafish in vivo, triggered by several inflammation and infection models that are known to trigger TNFɑ expression. These include injury, bacterial and parasitic infections. Zebrafish are a great animal model owing to the ease of genetic manipulation (e.g. knock-out by CRISPR-Cas9), available transgenic lines marking immune cells with fluorescent proteins, and in vivo live imaging offered by this animal model in combination with high-end microscopy. This will allow us to study in more details the specific role of TNFɑ and its receptors during inflammation.

Methodology

Depending on the length of the period, the work entails:

• Zebrafish breeding and embryo collection

In vivo imaging using stereo or confocal fluorescence microscopy

• Microinjection of CRISPR-Cas9 complexes in single cell-stage eggs

• DNA extraction and PCR analysis of selected immune genes (TNFα, TNFR, etc..)

• Image analysis and cell tracking

• Inflammation model:

- Tail injury and/or

- Microinjection of fluorescent bacteria and/or

- Microinjection of Parasites (trypanosomes)

The description is based on approximately 4-6 months lab work, and is a collaboration between the HMI and AFI group.

Requirements

A good background in Immunology, Molecular and Cellular Biology is required for this subject;

Contact person: Maria Forlenza, maria.forlenza@wur.nl