Reproductive Technology

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

Z. Smorg ,

R. Somski ,

L. Ktska-Ksikiewicz ,

D. Lipiski ,

A. Pawski ,

B. Ryska ,

M. Piekowski

Abstract
A novel technique of chimeric somatic cell cloning was applied for production of a transgenic rabbit (NT20). The karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of two-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos were either cultured in vitro up to blastocyst stage (Experiment I) or immediately after the cloning procedure were transferred into recipient-females (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 two-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 recipientfemales (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 the transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from two-cell stage rabbit embryo, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

Key words: Embryo • Assisted Reproductive Technology • Developmental biology • Early development

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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]