Transposon-Mediated Transgenesis in the Short-Lived African Killifish Nothobranchius furzeri, a Vertebrate Model for Aging

Abstract

The African killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in captivity. N. furzeri comprises several wild-derived strains with striking differences in longevity ranging from 3 to 9 months, which makes it a powerful vertebrate model for aging research. The short life cycle of N. furzeri should also facilitate studies on adult traits that are specific to vertebrates. Although progress has been made to generate a genetic linkage map and to start sequencing the genome of N. furzeri, tools to genetically manipulate this species of fish have not yet been developed. Here, we report the first establishment of transgenesis in N. furzeri. We use the Tol2 transposase system to generate transgenic N. furzeri that express green fluorescent protein driven by the Xenopus cytoskeletal actin promoter or the zebrafish heat-shock protein 70 promoter. We successfully generate stable transgenic lines of N. furzeri with germline transmission of integrated transgene. The development of transgenesis in N. furzeri provides a powerful tool to investigate the mechanisms underlying aging and longevity in a short-lived vertebrate model. Transgenesis in this fish will also facilitate the study of other phenotypes, including adult tissue regeneration and cognitive behavior.

Keywords: Nothobranchius furzeri; Tol2 transposase; aging model; killifish; longevity; transgenesis.

Figure 1

Transgenic constructs. Transgenic constructs containing two Tol2 recognition elements (1 and 2) flanking a cassette comprising a promoter driving the gfp reporter gene. Two promoters were used in this study: Xenopus borealis cytoskeletal actin (Cska) and zebrafish heat shock protein 70 (Hsp70). This cassette also contains the zebrafish cardiac myocyte light chain (Cmlc2) promoter driving the mCherry gene, although we have not analyzed expression of mCherry. The Tol2 transposase recognizes the Tol2 elements, excises the cassette, and integrates it into the host’s genome.

Figure 2

Microinjection plate and needle for transgenesis. (A) Microinjection plate cast using an ad hoc built plastic mold. Embryos selected for injection are aligned along the trenches and pierced with borosilicate needles. Up to 40 embryos could fit each trench. (B) Plastic mold design and measures. (C) Comparison between the microinjection needle for zebrafish (top) and N. furzeri (bottom). N. furzeri needles are sturdier than zebrafish needles, allowing better chorion piercing.

Figure 3

Expression of GFP in pCska-gfp Tol2 transgenic N. furzeri. GFP expression in live noninjected (top row), injected P0 fish (second row), and the F1 (third row), and F2 (bottom row) progeny of GFP-positive N. furzeri. Pictures were taken 12 days postfertilization for embryos, and 5 days posthatching for fry. Bright field and GFP images are shown for embryos. Scale bar: 1 mm. GFP, green fluorescent protein.

Figure 4

Expression of GFP in adult N. furzeri injected with pCska-gfp Tol2. GFP expression in noninjected P0 adult fish and in P0, F1, and F2 adult fish injected with the pCska-gfp Tol2 construct. The P0 and F1 fish are 3-month-old adults. The F2 fish is a 1-month-old adult. The GFP images for individual fish were digitally assembled from individual snapshots. Scale bar: 5 mm. GFP, green fluorescent protein.

Figure 5

Expression of GFP in N. furzeri injected with pHsp70-gfp Tol2. GFP expression in 12-day-old embryos, 5-day-old fry, and 3-month-old adult N. furzeri fish injected with the pHsp70-gfp Tol2 construct. GFP expression was observed even without heat-shocking the individuals. Bright field and GPF images are shown for embryos. Scale bar: 1 mm for embryos; 5 mm for fry and adult fish. GFP, green fluorescent protein.

Figure 6

Integration of the transgene into N. furzeri’s genome (A) PCR amplification of genomic DNA from transgenic fish using primers for gfp (upper panel) and igf1r (lower panel). The arrow indicates the amplified gfp band. F1+: F1 GFP-positive fish generated by crossing the P0 GFP-positive individual with a wild-type fish; F2+: F2 GFP-positive fish generated by intercrossing two F1 GFP-positive fish. n.i., noninjected control fish. (B) Southern blot on genomic DNA from transgenic fish using a probe for gfp. F1+: two F1 GFP-positive fish generated by crossing the P0 GFP-positive individual with a wild-type fish; F2+: two F2 GFP-positive fish generated by intercrossing two F1 GFP-positive fish. F1 and F2 fish with the same number (1 and 2) belong to the same family and, therefore, share the same band size (indicated by the arrows) for gfp. GFP, green fluorescent protein.