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Definition of three somatic adult cell nuclear transplant methods in zebrafish (Danio rerio): before, during and after egg activation by sperm fertilization

Published online by Cambridge University Press:  12 June 2009

M. Pérez-Camps ,
J. Cardona-Costa ,
M. Francisco-Simao  and
F. García-Ximénez
M. Pérez-Camps*
Affiliation:
Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Camino de Vera 14, 46071 Valencia, Spain. Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Spain.
J. Cardona-Costa
Affiliation:
Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Spain. Aquaculture and Environmental Research Group (ACUMA), Polytechnic University of Valencia, Spain.
M. Francisco-Simao
Affiliation:
Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Spain. Faculty of Agriculture Sciences, Agostinho Neto University, Angola.
F. García-Ximénez
Affiliation:
Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Spain.
*
All correspondence to: M. Pérez-Camps. Laboratory of Animal Reproduction and Biotechnology (LARB-UPV), Polytechnic University of Valencia, Camino de Vera 14, 46071 Valencia, Spain. Tel: +34 963879433. Fax: +34 963877439. e-mail: mipecam@dca.upv.es
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Summary

Zebrafish somatic nuclear transplant has only been attempted using preactivated eggs. In this work, three methods to carry out the nuclear transplant using adult cells before, during and after the egg activation/fertilization were developed in zebrafish with the aim to be used in reprogramming studies. The donor nucleus from somatic adult cells was inserted: (method A) in the central region of the egg and subsequently fertilized; (method B) in the incipient animal pole at the same time that the egg was fertilized; and (method C) in the completely defined animal pole after fertilization. Larval and adult specimens were obtained using the three methods. Technical aspects related to temperature conditions, media required, egg activation/fertilization, post-ovulatory time of the transplant, egg aging, place of the donor nucleus injection in each methodology are presented. In conclusion, the technical approach developed in this work can be used in reprogramming studies.

Keywords

Egg activationFertilizationMicroinjectionNuclear transplantZebrafish
Type
Research Article
Information
Zygote , Volume 18 , Issue 1 , February 2010 , pp. 33 - 39
Copyright
Copyright © Cambridge University Press 2009

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