A Simple and Efficient Microinjection Protocol for Making Transgenic Sticklebacks

Hosemann, Kim E.; Colosimo, Pamela F.; Summers, Brian R.; Kingsley, David M.

doi:10.1163/1568539042948097

Cited by 30 publications

(12 citation statements)

References 9 publications

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“…Analysis of EGFP expression revealed that all four larvae showed a mosaic expression pattern of EGFP as it has been frequently described for the founder generation of transgenic animals ( Fig. 3C,D ;Winkler et al, 1991;Hosemann et al, 2004;Cho et al, 2011). EGFP expression continued throughout adulthood as determined at the age of 7 months (not shown).…”

Section: Developmental Dynamicssupporting

confidence: 68%

A microinjection protocol for the generation of transgenic killifish (Species: Nothobranchius furzeri)

Hartmann

Englert

2012

Developmental Dynamics

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Background: A challenge in age research is the absence of short-lived vertebrate model organisms. The turquoise killifish Nothobranchius furzeri has an exceptionally short lifespan of 4-10 months depending on the strain. Thus, it possesses the shortest known maximum lifespan of a vertebrate species that can be bred in captivity. Results: Here we show the successful introduction of DNA and RNA molecules into the one-cell embryo of N. furzeri. For this purpose, we adapted existing microinjection protocols to inject through the remarkably thick and robust chorion of N. furzeri's eggs. The injected DNA transgene was integrated into the genome and transmitted to subsequent generations as indicated by the expression of the fluorophore enhanced green fluorescent protein (EGFP). Furthermore, we could confirm a special phase during embryonic development in which embryogenesis occurs within a re-aggregated mass of previously dispersed cells as it has been described for other related cyprinodont fish species. Conclusions: The transgenesis protocol described here provides a basis for a variety of genetic manipulations including overexpression of genes and determining their effects on lifespan and longevity. The feasibility to perform transgenesis is an important step to establish N. furzeri as a new model in age research.

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Section: Developmental Dynamicssupporting

confidence: 68%

A microinjection protocol for the generation of transgenic killifish (Species: Nothobranchius furzeri)

Hartmann

Englert

2012

Developmental Dynamics

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“…Injecting DNA into stickleback embryos typically results in far higher lethality than RNA alone. It is typical to see up to 50% (sometimes even more) lethality or malformation (see Figure 4D–F, I) after injecting Tol2 reporter constructs (similar to the previously described meganuclease method 21 ). However, the results vary widely based on the specific construct, the embryo morphology, and skill level.…”

Section: Representative Resultssupporting

confidence: 52%

“…A previously published protocol for generating transgenic sticklebacks using a meganuclease method 21 reported a 4–7% transgene germline transmission rate from F 0 founder fish. The Tol2 method reported here resulted in up to a 72% transgene germline transmission rate from F 0 founder fish (indicating multiple genomic integrations).…”

Section: Discussionmentioning

confidence: 99%

“…In addition to manipulating their coding sequences, the roles of candidate genes can be assessed by studying their cis -regulatory sequences and by functionally increasing, decreasing, or eliminating expression of the candidate gene. Microinjection and transgenesis methods in sticklebacks are well established 7,8,20 and were initially developed using a meganuclease-mediated method 21 first described in medaka 22 . The modified microinjection method presented here has been optimized for both Tol2-mediated transgenesis and recently developed genome editing reagents including TALENs and CRISPRs.…”

Section: Introductionmentioning

confidence: 99%

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Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Erickson

Ellis

Miller

2016

JoVE

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SHORT ABSTRACT Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback. LONG ABSTRACT The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

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“…edu) [5,7] has applied to stickleback techniques that were developed for research on skeletal development in mice [27]. These and other laboratories have developed methods for the analysis of gene expression during development [4,7,28,29], for generating transgenic stickleback [30], and have provided the first genomewide linkage map for stickleback [11].…”

mentioning

confidence: 99%

Evolution in parallel: new insights from a classic system

Foster¹

2004

Trends in Ecology & Evolution

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Neo-darwinists have long argued that parallel evolution, the repeated evolution of similar phenotypes in closely related lineages, is caused by the action of similar environments on alleles at many loci of small effect. A more controversial possibility is that the genetic architecture of traits initiates parallelism, sometimes through fixation of alleles of large effect. Recent research (by Cole et al., Colosimo et al., Cresko et al., and Shapiro et al.) offers the surprising insight that reduction in two armor traits of threespine stickleback is governed by independently segregating major loci as well as additional quantitative trait loci (QTL), and that alleles at the same major loci are associated with parallel phenotypes in globally distributed populations. This research suggests the emergence of a new and exciting vertebrate model system for evolutionary genetics.

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A Simple and Efficient Microinjection Protocol for Making Transgenic Sticklebacks

Cited by 30 publications

References 9 publications

A microinjection protocol for the generation of transgenic killifish (Species: Nothobranchius furzeri)

A microinjection protocol for the generation of transgenic killifish (Species: Nothobranchius furzeri)

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

Evolution in parallel: new insights from a classic system

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