Methods for generating and colonizing gnotobiotic zebrafish

Abstract

Vertebrates are colonized at birth by complex and dynamic communities of microorganisms that can contribute significantly to host health and disease. The ability to raise animals in the absence of microorganisms has been a powerful tool for elucidating the relationships between animal hosts and their microbial residents. The optical transparency of the developing zebrafish and relative ease of generating germ-free (GF) zebrafish make it an attractive model organism for gnotobiotic research. Here we provide a protocol for generating zebrafish embryos; deriving and rearing GF zebrafish; and colonizing zebrafish with microorganisms. Using these methods, we typically obtain 80-90% sterility rates in our GF derivations with 90% survival in GF animals and 50-90% survival in colonized animals through larval stages. Obtaining embryos for derivation requires approximately 1-2 h, with a 3- to 8-h incubation period before derivation. Derivation of GF animals takes 1-1.5 h, and daily maintenance requires 1-2 h.

Figure 1

Schematic diagram of isolator equipped for gnotobiotic zebrafish husbandry.

Figure 2. Schematic diagram of zebrafish laparotomy

Incisions to be made are enumerated and shown in blue.

Figure 3. Overview of gnotobiotic zebrafish husbandry

Corresponding procedure steps (blue text) and timing requirements (green text) are indicated.

Figure 4. Comparison of colonization methods in zebrafish

Fish are colonized 3 days post fertilization (dpf) because this is the stage at which CONV-R animals normally hatch from their chorions and are colonized. Timing of colonization and analysis can be defined by user. Blue indicates fish in the GF state, green indicates fish colonized with the microbial community present in conventionalized animal, and red indicates fish colonized with a microbe of choice. CONVD, CONV-R, and monoassociated fish usually possess 10⁴–10⁵ CFU/gut at 6dpf.