JARB Journal of Animal Reproduction and Biotehnology

OPEN ACCESS pISSN: 2671-4639
eISSN: 2671-4663

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Journal of Embryo Transfer 1998; 13(3): 219-226

Published online December 1, 1998

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

활성화된 수핵란을 이용한 핵이식기법의 개선

윤희준, 이효종, 최상용, 박충생

경상대학교 축산진흥연구소

Abstract

Enucleation of oocytes is an important limiting step for embryo cloning. We propose an enucleation technique based on the removal of chromatin after oocyte activation by aspirating the second polar body containing complemented chromatin. In a preliminary experiment to determine an optimal age of oocytes enucleation in rabbits, oocytes were enucleated at 15~20 hours post hCG. Recently ovulated oocytes were enucleated at a higher rate than aged oocytes. Microsurgical removal of the complemented chromatin in the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes(96.8% versus 70.4%; P〈0.05). Moreover, compared with a nuclear transplantation protocol based on enucleation of metaphase-arrested oocytes and preactivated oocytes followed by treatment with 5 $mu$M ionomycin for 5 min and 2 mM DMAP for 1 hr, there was no significant difference in the rate of blastocyst development. The ease with which modified technique can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

Keywords: enucleation, oocyte activation, ionomycin, DMAP, nuclear transfer, rabbit

Article

Journal of Embryo Transfer 1998; 13(3): 219-226

Published online December 1, 1998

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

활성화된 수핵란을 이용한 핵이식기법의 개선

윤희준, 이효종, 최상용, 박충생

경상대학교 축산진흥연구소, 경상대학교 수의과대학 수의학과, 경상대학교 수의과대학 수의학과, 경상대학교 농과대학 축산학과

활성화된 수핵란을 이용한 핵이식기법의 개선

윤희준, 이효종, 최상용, 박충생

경상대학교 축산진흥연구소

Abstract

Enucleation of oocytes is an important limiting step for embryo cloning. We propose an enucleation technique based on the removal of chromatin after oocyte activation by aspirating the second polar body containing complemented chromatin. In a preliminary experiment to determine an optimal age of oocytes enucleation in rabbits, oocytes were enucleated at 15~20 hours post hCG. Recently ovulated oocytes were enucleated at a higher rate than aged oocytes. Microsurgical removal of the complemented chromatin in the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes(96.8% versus 70.4%; P〈0.05). Moreover, compared with a nuclear transplantation protocol based on enucleation of metaphase-arrested oocytes and preactivated oocytes followed by treatment with 5 $mu$M ionomycin for 5 min and 2 mM DMAP for 1 hr, there was no significant difference in the rate of blastocyst development. The ease with which modified technique can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

Keywords: enucleation, oocyte activation, ionomycin, DMAP, nuclear transfer, rabbit