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G3 (Bethesda)
2011 Dec 01;17:531-8. doi: 10.1534/g3.111.001271.
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Transposon-Mediated Transgenesis in the Short-Lived African Killifish Nothobranchius furzeri, a Vertebrate Model for Aging.
Valenzano DR
,
Sharp S
,
Brunet A
.
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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.
Figure 1â. 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 hostâs genome.
Figure 2â. 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 3â. 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 4â. 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 5â. 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 6â. Integration of the transgene into N. furzeriâs 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.
Chan,
Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer.
2010, Pubmed
Chan,
Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer.
2010,
Pubmed
Davidson,
Efficient gene delivery and gene expression in zebrafish using the Sleeping Beauty transposon.
2003,
Pubmed
Di Cicco,
The short-lived annual fish Nothobranchius furzeri shows a typical teleost aging process reinforced by high incidence of age-dependent neoplasias.
2011,
Pubmed
Egami,
Life span data for the small fish, Oryzias latipes.
1969,
Pubmed
Genade,
Annual fishes of the genus Nothobranchius as a model system for aging research.
2005,
Pubmed
Grabher,
Meganuclease and transposon mediated transgenesis in medaka.
2007,
Pubmed
Guyomard,
Integration and germ line transmission of foreign genes microinjected into fertilized trout eggs.
1989,
Pubmed
Hamlet,
Tol2 transposon-mediated transgenesis in Xenopus tropicalis.
2006,
Pubmed
,
Xenbase
Hartmann,
Telomeres shorten while Tert expression increases during ageing of the short-lived fish Nothobranchius furzeri.
2009,
Pubmed
Inglima,
Reversible stage-specific embryonic inhibition mediated by the presence of adults in the annual fish Nothobranchius guentheri.
1981,
Pubmed
Iwamatsu,
Stages of normal development in the medaka Oryzias latipes.
2004,
Pubmed
Kawakami,
A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.
2004,
Pubmed
Kawakami,
Tol2: a versatile gene transfer vector in vertebrates.
2007,
Pubmed
,
Xenbase
Kawakami,
Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage.
2000,
Pubmed
Kawakami,
Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element.
2004,
Pubmed
Koga,
Germline transgenesis of zebrafish using the medaka Tol1 transposon system.
2008,
Pubmed
Lakin,
Determination of the sequence requirements for the expression of a Xenopus borealis embryonic/larval skeletal actin gene.
1993,
Pubmed
,
Xenbase
Levels,
Oxygen consumption during embryonic development of the annual fish Nothobranchius korthausae with special reference to diapause.
1986,
Pubmed
Lu,
Integration, expression and germ-line transmission of foreign growth hormone genes in medaka (Oryzias latipes).
1992,
Pubmed
Markofsky,
The effects of temperature and season of collection on the onset and duration of diapause in embryos of the annual fish Nothobranchius guentheri.
1977,
Pubmed
Martínez,
Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone.
1996,
Pubmed
Matias,
The stage-dependent resistance of the chorion to external chemical damage and its relationship to embryonic diapause in the annual fish, Nothobranchius guentheri.
1984,
Pubmed
Ni,
Transposon tools hopping in vertebrates.
2008,
Pubmed
Podrabsky,
The bioenergetics of embryonic diapause in an annual killifish, austrofundulus limnaeus.
1999,
Pubmed
Podrabsky,
An inducible 70 kDa-class heat shock protein is constitutively expressed during early development and diapause in the annual killifish Austrofundulus limnaeus.
2007,
Pubmed
Podrabsky,
Survival of water stress in annual fish embryos: dehydration avoidance and egg envelope amyloid fibers.
2001,
Pubmed
Reichwald,
High tandem repeat content in the genome of the short-lived annual fish Nothobranchius furzeri: a new vertebrate model for aging research.
2009,
Pubmed
Stuart,
Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos.
1988,
Pubmed
Suster,
Transgenesis in zebrafish with the tol2 transposon system.
2009,
Pubmed
SWARUP,
Stages in the development of the stickleback Gasterosteus aculeatus (L.).
1958,
Pubmed
Takeda,
The art of medaka genetics and genomics: what makes them so unique?
2010,
Pubmed
Terzibasi,
Effects of dietary restriction on mortality and age-related phenotypes in the short-lived fish Nothobranchius furzeri.
2009,
Pubmed
Terzibasi,
Large differences in aging phenotype between strains of the short-lived annual fish Nothobranchius furzeri.
2008,
Pubmed
Thermes,
I-SceI meganuclease mediates highly efficient transgenesis in fish.
2002,
Pubmed
Urasaki,
Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition.
2006,
Pubmed
Valenzano,
Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri.
2006,
Pubmed
Valenzano,
Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate.
2006,
Pubmed
Valenzano,
Mapping loci associated with tail color and sex determination in the short-lived fish Nothobranchius furzeri.
2009,
Pubmed
Yaskowiak,
Characterization and multi-generational stability of the growth hormone transgene (EO-1alpha) responsible for enhanced growth rates in Atlantic Salmon.
2006,
Pubmed
