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This Article Full Text Full Text (PDF) All Versions of this Article: 68/6/2013    most recent biolreprod.102.010066v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Bordignon, V. Articles by Smith, L. C. Search for Related Content PubMed PubMed Citation Articles by Bordignon, V. Articles by Smith, L. C. Agricola Articles by Bordignon, V. Articles by Smith, L. C.

Transgene Expression of Green Fluorescent Protein and Germ Line Transmission in Cloned Calves Derived from In Vitro-Transfected Somatic Cells¹

Centre de recherche en reproduction animal,³ Département de sciences cliniques,⁴ Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec, Canada J2S 7C6 Nexia Biotechnologies, Inc.,⁵ Vaudreuil-Dorion, Quebec, Canada J7V 8P5

In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP)-reporter transgene and used as nuclear donor cells in oocyte reconstructions. Because cell synchronization protocols are less effective after transfection, activated oocytes may be more suitable as hosts for nuclear transfer. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either before (metaphase) or after (telophase) activation. Expression of GFP was examined during early embryogenesis, in tissues of cloned calves, and again during embryogenesis, after passage through germ line using semen from the transgenic cloned offspring. Regardless of the kind of host cytoplasm used, GFP became detectable at the 8- to 16-cell stage, approximately 80 h after reconstruction, and remained positive at all later stages. After birth, although cloned calves obtained through both procedures expressed GFP in all tissues examined, expression levels varied both between tissues and between cells within the same tissue, indicating a partial shutdown of GFP expression during cellular differentiation. Moreover, nonexpressing fibroblasts derived from transgenic offspring were unable to direct GFP expression after nuclear transfer and development to the blastocyst stage, suggesting an irreversible silencing of transgenes. Nonetheless, GFP was expressed in approximately half the blastocysts obtained with sperm from a transgenic clone, confirming transmission of the transgene through the germ line.

¹ Supported by Conseil de recherche en pêche et agroalimentaire du Québec (CORPAQ) and the National Science and Engineering Council (NSERC) of Canada.

² Correspondence: Lawrence C. Smith, University of Montreal, CRRA/Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte, Ste-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; smithl{at}medvet.umontreal.ca