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ISSN 2052-1472 (online)

Reproduction Abstracts (2014) 1 P081 | DOI: 10.1530/repabs.1.P081

Establishment of efficient system in the DNA microinjection into porcine in vitro embryos

Don Buddika Oshadi Malaweera1, Sisitha Ramachandra1, Ghangyong Kim1, Seokjoong Kim2 & Jongki Cho1


1Chungnam National University, Daejeon, Republic of Korea; 2Toolgen, Seoul, Republic of Korea.


To investigate the efficient laboratory techniques for the injection of DNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and IVF embryos to explore the injection time, volume and concentration, for the efficient blastocyst production. In experiment 1, to investigate injection time, compared four different time durations (2, 4, 6 and 8 h) after post activation and 6 h of co-incubation with sperms. There were no significant difference (P<0.05) between four groups regarding the percentage of cleavages. However there were significant difference (P<0.05) in development (4.4, 8.9, 3.9, 0.6%), GFP expression (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 h compared with another three groups. IVF embryos after 2 and 4 h were expressed GFP significantly higher than rest of two groups. In experiment 2, two concentrations of 20 and 50 ng/μl were injected to the embryos and observed blastocysts after 7 days of incubation. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts rate (13.0 vs 11.1%) and GFP expression (5.7 vs 0.0%) in 20 ng/μl. In IVF embryos, only 20 ng/μl injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development. In experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos. Out of three groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05).

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