Name: Obtaining Wildtype and Transgenic Zebrafish Larvae and WildType Whole Larvae Dissociation
Uploaded: 2023-11-10T14:00:00
Description: Watch this Scientific Journal Video about Obtaining Wildtype and Transgenic Zebrafish Larvae and WildType Whole Larvae Dissociation at JoVE.com

obtaining wildtype and transgenic zebrafish larvae and wildtype whole larvae dissociation

Behandler tarmceller fra sebrafisklarver for encellet RNA-sekvensering

The gastrointestinal (GI) tract is a complex system that plays a vital role in overall health and well-being. It is responsible for the digestion and absorption of nutrients, as well as the elimination of waste products1,2. The GI tract is composed of multiple cell types, including epithelial cells, smooth muscle cells, immune cells, and the enteric nervous system (ENS), which communicate closely together to regulate and maintain proper intestinal function3,4,5. Defects in the development of the GI tract can have far-reaching effects on various aspects such as nutrient absorption, microbiota composition, the gut-brain axis, and the ENS, leading to several enteric neuromuscular disorders, such as Hirschsprung disease and Chronic intestinal pseudo-obstruction6,7. These disorders are characterized by severe gut dysmotility caused by alterations in various key cells, such as the interstitial cells of Cajal, smooth muscle cells, and the ENS6,8,9. However, the molecular mechanisms underlying GI development and dysfunction are still poorly understood.
The zebrafish is a valuable model organism for studying GI development and dysfunction due to its rapid embryonic development, transparency during embryonic and larval stages, and genetic tractability10,11,12,13,14. Numerous transgenic zebrafish lines expressing fluorescent proteins are available. An example of such a line is the tg(phox2bb:GFP) zebrafish, commonly used to study the ENS, as all phox2bb+ cells, including enteric neurons, are labeled15,16. Here, using the tg(phox2bb:GFP) zebrafish line, we present a method for intestinal isolation of 5 days post fertilization (dpf) larvae for single-cell RNA sequencing (scRNA-seq) analysis (Figure 1).

Forfatter Spotlight: Isolering og analyse av tarmceller fra sebrafisklarver for å undersøke transkriptomiske aspekter ved gastrointestinal utvikling 

Tarmisolasjon fra sebrafisklarver for enkeltcelle RNA-sekvensering

Our research aims to investigate the transcriptomic changes occurring during gastrointestinal development and dysfunction in zebrafish larvae. Here, we provide a new protocol that allows isolation of different cell types within the zebrafish intestine, which has potential applications in understanding gastrointestinal health and disease. In recent years, there has been growing interest in exploring the interaction between the enteric nervous system and other intestinal cell types during development and disease.Understanding these complex dynamics using advanced techniques like single-cell RNA sequencing, holds the potential to reveal critical insights into enteric neuromyopathies, leading to more precise diagnostics and treatments. Using this protocol, we successfully identified various intestinal cell types, including epithelial, stromal, bloods, muscle, immune cells, and even enteric neurons and glia, which have not been captured before at this amount using similar approaches. Thus, we provide a reliable technique to study the gastrointestinal composition during development and dysfunction.

Watch this Scientific Journal Video about Obtaining Wildtype and Transgenic Zebrafish Larvae and WildType Whole Larvae Dissociation at JoVE.com

Obtaining Wildtype and Transgenic Zebrafish Larvae and WildType Whole Larvae Dissociation  

Innhenting av villtype og transgene sebrafisklarver og vilttype hele larver dissosiasjon

Begynn med å skaffe villtype og transgene sebrafiskegg fra det respektive avlsoppsettet. Ta en 15-centimeter petriskål som inneholder HEPES-bufret E3 medium eller E3. Og samle riktig mengde befruktede egg fra ønsket avlsoppsett for sebrafisk. Inkuber de befruktede eggene i E3 ved 28 grader Celsius med en passende lys og mørk syklus.På en dag etter befruktning eller dpf, fjern eventuelle ubefruktede egg og la de befruktede utvikle seg til fem dpf. Velg de transgene sebrafisklarvene på en dpf. Før du fortsetter for hele larver dissosiasjon, bedøve de fem dpf wildtype larver med 0,016% Tricaine, og overføre 30 bedøvede larver i en petriskål som inneholder en milliliter 10x trypsin-EDTA løsning.Deretter, ved hjelp av et barberblad, hakk larvene i små biter i petriskålen. Overfør de hakkede larvene til et mikrosentrifugerør med to milliliter 10x trypsin-EDTA-løsning, og la røret ligge på is i tre timer. Pipetter innholdet opp og ned med en P1000-pipette hver time for å stimulere dissosiasjon.

Research