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An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes).

ABSTRACT: BACKGROUND:The CRISPR/Cas system is a powerful genome editing tool that enables targeted genome modifications in various organisms. In medaka (Oryzias latipes), targeted mutagenesis with small insertions and deletions using this system have become a robust technique and are now widely used. However, to date there have been only a small number of reports on targeted gene integration using this system. We thus sought in the present study to identify factors that enhance the efficiency of targeted gene integration events in medaka. RESULTS:We show that longer homology arms (ca. 500 bp) and linearization of circular donor plasmids by cleavage with bait sequences enhances the efficiency of targeted integration of plasmids in embryos. A new bait sequence, BaitD, which we designed and selected by in silico screening, achieved the highest efficiency of the targeted gene integration in vivo. Using this system, donor plasmids integrated precisely at target sites and were efficiently transmitted to progeny. We also report that the genotype of F2 siblings, obtained by mating of individuals harboring two different colors of fluorescent protein genes (e.g. GFP and RFP) in the same locus, can be easily and rapidly determined non-invasively by visual observations alone. CONCLUSION:We report that the efficiency of targeted gene integration can be enhanced by using donor vectors with longer homologous arms and linearization using a highly active bait system in medaka. These findings may contribute to the establishment of more efficient systems for targeted gene integration in medaka and other fish species.

SUBMITTER: Murakami Y

PROVIDER: S-EPMC5500998 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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An efficient system for homology-dependent targeted gene integration in medaka (<i>Oryzias latipes</i>).

Murakami Yu Y Ansai Satoshi S Yonemura Akari A Kinoshita Masato M

Zoological letters 20170706

<h4>Background</h4>The CRISPR/Cas system is a powerful genome editing tool that enables targeted genome modifications in various organisms. In medaka (<i>Oryzias latipes</i>), targeted mutagenesis with small insertions and deletions using this system have become a robust technique and are now widely used. However, to date there have been only a small number of reports on targeted gene integration using this system. We thus sought in the present study to identify factors that enhance the efficien ...[more]

PMID: 28694996

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Project description:BackgroundPrimordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. Two modes of germ line specification have been described: the inheritance of asymmetrically-localized maternally provided cytoplasmic determinants and the induction of the PGC fate by other cell types. PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression--the vasa homologue in another teleost, medaka (Oryzias latipes). Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species.ResultsIn this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm.ConclusionTaken together, these results are consistent with the idea that in medaka the inheritance of maternally provided asymmetrically-localized cytoplasmic determinants directs cells to assume the germ line fate similar to zebrafish PGCs.

Dataset Information

An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes).

Publications

An efficient system for homology-dependent targeted gene integration in medaka (<i>Oryzias latipes</i>).

Similar Datasets

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