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. 2015;61(3):241-4.
doi: 10.1262/jrd.2014-157. Epub 2015 Feb 10.

A Hyperactive piggyBac Transposon System Is an Easy-To-Implement Method for Introducing Foreign Genes Into Mouse Preimplantation Embryos

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Free PMC article

A Hyperactive piggyBac Transposon System Is an Easy-To-Implement Method for Introducing Foreign Genes Into Mouse Preimplantation Embryos

Shinnosuke Suzuki et al. J Reprod Dev. .
Free PMC article

Abstract

Transgenic mice are important tools for genetic analysis. A current prominent method for producing transgenic mice involves pronuclear microinjection into 1-cell embryos. However, the total transgenic efficiency obtained using this method is less than 10%. Here, we demonstrate that highly efficient transgenesis in mice can be achieved by cytoplasmic microinjection using a hyperactive piggyBac system. In embryos in which hyPBase mRNA and pPB-CAG-TagRFP DNA were co-injected into the cytoplasm, TagRFP fluorescence was observed after the 2-cell stage; when 30 ng/µl pPB-CAG-TagRFP DNA and 30 ng/µl hyPBase mRNA were co-injected, 94.4% of blastocysts were TagRFP positive. Furthermore, a high concentration of hyPBase mRNA resulted in creation of mosaic embryos in which the TagRFP signals partially disappeared. However, suitable concentrations of injected DNA and hyPBase mRNA produced embryos in which almost all blastomeres were TagRFP positive. Thus, the hyperactive piggyBac transposon system is an easy-to-implement and highly effective method that can contribute to production of transgenic mice.

Figures

Fig. 1.
Fig. 1.
A schematic illustration of microinjection into the cytoplasm of 1-cell embryos and target DNA integration into genomic DNA mediated by hyPBase proteins.
Fig. 2.
Fig. 2.
Fluorescence of TagRFP in embryos co-injected with pPB-CAG-TagRFP DNA and hyPBase mRNA. (A) Representative photos showing TagRFP fluorescence in embryos co-injected with pPB-CAG-TagRFP DNA (30 ng/µl) and hyPBase mRNA (0, 10, 30, 50 and 100 ng/µl) at the blastocyst stage. Embryos were photographed 108 h after in vitro fertilization. Scale bar, 100 µm. (B) Representative photos showing TagRFP fluorescence in embryos co-injected with pPB-CAG-TagRFP DNA (30 ng/µl) and hyPBase mRNA (0, 10, 30, 50 and 100 ng/µl) at the 2-cell stage. Embryos were photographed 38 h after in vitro fertilization. Scale bar, 100 µm.
Fig. 3.
Fig. 3.
Fluorescence of TagRFP in embryos co-injected with pPB-CAG-TagRFP DNA and hyPBase mRNA at the blastocyst stage. Detection of TagRFP fluorescence in embryos co-injected with pPB-CAG-TagRFP DNA (30 ng/µl) and hyPBase mRNA (0, 10, 30, 50 and 100 ng/µl) (red, Tag-RFP; blue, chromatin). Scale bars, 100 µm.

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