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A hyperactive piggyBac transposon system is an easy-to-implement method for introducing foreign genes into mouse preimplantation embryos.

Suzuki S, Tsukiyama T, Kaneko T, Imai H, Minami N - J. Reprod. Dev. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.

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.

No MeSH data available.


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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fig_003: 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.

Mentions: a,b Values with different superscripts within the same column are significantly different (P < 0.05).


A hyperactive piggyBac transposon system is an easy-to-implement method for introducing foreign genes into mouse preimplantation embryos.

Suzuki S, Tsukiyama T, Kaneko T, Imai H, Minami N - J. Reprod. Dev. (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4498375&req=5

fig_003: 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.
Mentions: a,b Values with different superscripts within the same column are significantly different (P < 0.05).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.

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.

No MeSH data available.