Dataset Information

A Collection of Transgenic Medaka Strains for Efficient Site-Directed Transgenesis Mediated by phiC31 Integrase.

ABSTRACT: Genetic analysis is facilitated by the efficient production of transgenic strains expressing a DNA of interest as a single copy at a designated chromosomal location. However, technical progress toward this goal in medaka fish (Oryzias latipes), a vertebrate model organism, has been slow. It is well known that phiC31 integrase enables efficient site-directed transgenesis by catalyzing the recombination of an attP DNA motif in a host genome with an attB motif in a targeting vector. This system was pioneered in medaka using the Sleeping Beauty transposon system, and the attP site was established at three chromosomal locations. However, this number appeared insufficient with regard to genetic linkage between the attP-landing site and a genetically modified locus of interest. Here, to establish a collection of transgenic strains of medaka, we introduced an attP motif into the medaka genome using the Ac/Ds maize transposon system and established 12 independent transgenic strains harboring a single copy of the attP motif in at least 11 of the 24 medaka chromosomes. We designed an attB-targeting vector that was integrated efficiently and precisely into the attP-landing site, and with which the DNA of interest was efficiently transmitted to germline cells. Extraneous sequences in the integrants derived from the bacterial backbone of the attB-targeting vector as well as a transgenic fluorescence marker present in the attP-landing site were removable through flippase-mediated recombination. Further, an advanced targeting vector with a heart-specific recombination marker served as a useful tool for easily screening phiC31 integrase-mediated recombinant G0 embryos, leading to the efficient establishment of transgenic strains. Thus, our resources advance genetic research in medaka.

SUBMITTER: Ishikawa T

PROVIDER: S-EPMC6071608 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

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A Collection of Transgenic Medaka Strains for Efficient Site-Directed Transgenesis Mediated by phiC31 Integrase.

Ishikawa Tokiro T Ansai Satoshi S Kinoshita Masato M Mori Kazutoshi K

G3 (Bethesda, Md.) 20180731 8

Genetic analysis is facilitated by the efficient production of transgenic strains expressing a DNA of interest as a single copy at a designated chromosomal location. However, technical progress toward this goal in medaka fish (<i>Oryzias latipes</i>), a vertebrate model organism, has been slow. It is well known that phiC31 integrase enables efficient site-directed transgenesis by catalyzing the recombination of an attP DNA motif in a host genome with an attB motif in a targeting vector. This sys ...[more]

PMID: 29848622

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Project description:Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified. The improved technology we describe here will facilitate comparative studies of effector transgenes, allowing informed choices to be made that potentially lead to transmission blockade.

Project description:BackgroundTransgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI) for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells.MethodsAccording to this system, a "docking-site" was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an "incoming" vector containing the gene of interest was specifically inserted in the docking-site using PhiC31.ResultsUsing the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate format.ConclusionsThe novel PhiC31-IMSI system described in this study represents a powerful tool that can facilitate the characterization of cancer-related genes.

Dataset Information

A Collection of Transgenic Medaka Strains for Efficient Site-Directed Transgenesis Mediated by phiC31 Integrase.

Publications

A Collection of Transgenic Medaka Strains for Efficient Site-Directed Transgenesis Mediated by phiC31 Integrase.

Similar Datasets

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