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Generation of Transgenic Rabbits by the Novel Technique of Chimeric Somatic Cell Cloning¹

Z. Smorg ³

R. Somski ⁴⁵ 

L. Ktska-Ksikiewicz ³

D. Lipiski ⁵

A. Pawski ⁵

B. Ryska ³

M. Piekowski ⁶

Department of Animal Reproduction Biotechnology,³ National Research Institute of Animal Production, 32–083 Balice/Kraków, Poland Department of Biochemistry and Biotechnology,⁴ Agricultural University in Pozna, 60–637 Pozna, Poland Institute of Human Genetics,⁵ Polish Academy of Sciences, 60–479 Pozna, Poland Allergic Diseases, Asthma and Immunology Clinic,⁶ P.C., Knoxville, Tennessee 37919

ABSTRACT

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

embryo, assisted reproductive technology, developmental biology, early development

¹ Supported by the State Committee for Scientific Research as the Solicited Project nos. PBZ-KBN-048/P05/2001/08 and PBZ-KBN-048/P05/2001/04 from 2003–2005 and by the Allergic Diseases, Asthma and Immunology Clinic, P.C., Knoxville, TN.

² Correspondence. FAX: 4812 285 51 62; mskrzysz{at}izoo.krakow.pl

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				P. Greda, J. Karasiewicz, and J. A Modlinski Mouse zygotes as recipients in embryo cloning. Reproduction, November 1, 2006; 132(5): 741 - 748. [Abstract] [Full Text] [PDF]