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Reproduction Abstracts (2014) 1 P078 | DOI: 10.1530/repabs.1.P078

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The effect of cRNA concentration of artificial nuclease microinjected cytoplasmically to pronuclear porcine embryos on survival and development in vitro

Maki Kamoshita1, Tsubasa Kato1, Eri Sagara2, S Hisamatsu3, M Sakaue2, Tetsushi Sakuma4, Takashi Yamamoto4, Junya Ito5 & Naomi Kashiwazaki5

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1Graduate School of Veterinary Sciences, Azabu University, Kanagawa, Japan; 2School of Veterinary Medicine, Azabu University, Kanagawa, Japan; 3Faculty of Life and Environmental Sciences, Azabu University, Kanagawa, Japan; 4Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; 5Azabu University, Sagamihara-shi, Japan.


Although GH receptor (GHR) gene deletion causes a hereditary dwarfism and also shows a longer lifespan, the fact was shown only in mice. Because it was difficult to generate knockout (KO) animals other than mice. Recently, the generation of KO animals became possible without difficulty due to advances in genome editing technologies such as transcription activator-like effector nuclease (TALEN). In the present study, we examined the effect of cRNA concentration microinjected cytoplasmically to pronuclear porcine embryos on survival and development of embryos in vitro. Porcine follicular oocytes were matured and then fertilized (IVF) in vitro. At 10 h after IVF, oocytes were centrifuged to visualize the pronuclei. Embryos with two or three pronuclei were injected cytoplasmically with 2 or 100 μg/ml of GHR TALEN cRNA. TALEN cDNAs for GHR were transcribed in vitro using mMESSAGE mMACHINE T7 Ultra kit and the products were diluted with RNase free water. After microinjection, embryos were cultured and observed survival at 15 min after microinjection and development at 48 and 134 h after culture. There were no significant differences in survival rates between 2 and 100 μg/ml groups (80.3 and 87.3%) (P>0.05). The rates of cleavage and blastocyst formation in 2 μg/ml group (50.9 and 35.1%) were significantly higher (P<0.05) than those in 100 μg/ml group (25.8 and 16.1%). The results suggested that 100 μg/ml of GHR TALEN cRNA may negatively affect development of porcine embryos. On the basis of the results, further study is required to generate GHR gene KO pigs.

Volume 1

World Congress of Reproductive Biology 2014

Edinburgh, UK
02 Sep 2014 - 04 Sep 2014

World Congress of Reproductive Biology 

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