Method for somatic cell nuclear transfer in zebrafish.

This chapter presents a detailed methodology for somatic cell nuclear transfer-cloning of zebrafish. We aim to place the reader in a virtual lab experience to assist acquisition of the technical skills required for reproducing the published protocol. All materials, including catalog numbers for reagents and techniques for their preparation, are provided. Our protocols describe laser inactivation of egg chromosomes, the transfer of a cell through the oocyte micropyle, and spontaneous activation of the reconstructed embryo. High-quality eggs are the key to cloning success, and Chinook salmon ovarian fluid is indispensable for keeping eggs arrested at the metaphase of meiosis II. This protocol continues to be refined by our laboratory. However, naive investigators should be able to apply it in its present form to generate cloned zebrafish.

[1]  J. Steibel,et al.  Characterization and in vitro control of MPF activity in zebrafish eggs. , 2009, Zebrafish.

[2]  S. Sivasubbu,et al.  Enhancer trapping in zebrafish using the Sleeping Beauty transposon , 2004, BMC Genomics.

[3]  Nancy Hopkins,et al.  Identification of 315 genes essential for early zebrafish development. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Ekker,et al.  Effective targeted gene ‘knockdown’ in zebrafish , 2000, Nature Genetics.

[5]  Chia-Cheng Chang,et al.  Somatic cell nuclear transfer in zebrafish , 2009, Nature Methods.

[6]  Ralf Dahm,et al.  Zebrafish: A Practical Approach. Edited by C. NÜSSLEIN-VOLHARD and R. DAHM. Oxford University Press. 2002. 322 pages. ISBN 0 19 963808 X. Price £40.00 (paperback). ISBN 0 19 963809 8. Price £80.00 (hardback). , 2003 .

[7]  A. Amores,et al.  Banded chromosomes and the zebrafish karyotype. , 1999, Methods in cell biology.

[8]  Dan Cherubin.  Eggs , 2006, The Essential Guide to Rockpooling.

[9]  T. Hocking,et al.  Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases , 2008, Nature Biotechnology.

[10]  I. Wilmut,et al.  Viable offspring derived from fetal and adult mammalian cells , 1997, Nature.

[11]  A. Schier,et al.  Efficient recovery of ENU-induced mutations from the zebrafish germline. , 1994, Genetics.

[12]  M. Noyes,et al.  Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases , 2008, Nature Biotechnology.

[13]  G. Streisinger,et al.  Induction of recessive lethal and specific locus mutations in the zebrafish with ethyl nitrosourea. , 1992, Genetical research.

[14]  Akihiro Urasaki,et al.  Insertional mutagenesis by the Tol2 transposon-mediated enhancer trap approach generated mutations in two developmental genes: tcf7 and synembryn-like , 2007, Development.

[15]  Ki-Young Lee,et al.  Cloned zebrafish by nuclear transfer from long-term-cultured cells , 2002, Nature Biotechnology.

[16]  Koichi Kawakami,et al.  The influence of donor nucleus source on the outcome of zebrafish somatic cell nuclear transfer. , 2010, The International journal of developmental biology.