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Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression.

ABSTRACT: Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo 'gain-of-function' and 'loss-of-function' studies.

SUBMITTER: Ohtsuka M

PROVIDER: S-EPMC3001095 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression.

Ohtsuka Masato M Ogiwara Sanae S Miura Hiromi H Mizutani Akiko A Warita Takayuki T Sato Masahiro M Imai Kenji K Hozumi Katsuto K Sato Takehito T Tanaka Masafumi M Kimura Minoru M Inoko Hidetoshi H

Nucleic acids research 20100929 22

Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated ...[more]

PMID: 20880997

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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

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression.

Publications

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression.

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