80 DEVELOPMENTAL POTENTIAL OF BOVINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM FETAL FIBROBLASTS TRANSDUCED WITH SIMIAN VIRUS 40 LARGE T ANTIGEN
V. Zakhartchenko A , F. Yang A , B. Vogg B , A. Pfeifer B and E. Wolf AA Department of Molecular Animal Breeding and Biotechnology, University of Munich, Munich, Germany email: fk_yang@yahoo.com;
B Department of Molecular Pharmacology, University of Munich, Munich, Germany.
Reproduction, Fertility and Development 16(2) 162-162 https://doi.org/10.1071/RDv16n1Ab80
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
Abstract
Primary cultured cells with limited lifespan are generally used as donors for nuclear transfer (NT). Immortalization or even extension of their proliferative capacity would provide an additional time-window for various genetic manipulations prior to NT. Previously, we reported that a spontaneously immortalized mammary epithelial cell line failed to support development of reconstructed embryos into blastocysts [Zakhartchenko et al., 1999 Mol. Reprod. Dev. 54, 264–272]. In contrast, telomerase-immortalized sheep fibroblasts could be substantially reprogrammed, although they were not fully competent for NT since no fetuses survived beyond 40 days of development [Cui et al., 2003 Biol. Reprod. 69, 15–21]. Simian virus 40 large T antigen (SV40Tag), a viral oncoprotein, is known to immortalize human diploid fibroblasts by soaking up the cellular negative growth regulators, pRb, p53, and some related factors to enable cells to grow continuously [Kim et al., 2001 Exp. Mol. Med. 33, 293–298]. In this study we examined the developmental competence of bovine NT embryos derived from SV40Tag-transduced fetal fibroblasts. Primary fetal fibroblasts (BFF) obtained from a 49-day-old fetus were first transduced with the green fluorescent protein (GFP) gene and then with SV40Tag using a replication-defective retrovirus. GFP-SV40Tag-positive cells (BFF-GFP-Tag), which can proliferate over 50 passages, were cultured until confluence and then used for nuclear transfer at passages 27–30. As control, confluent GFP-positive cells (BFF-GFP) and BFF, which stopped proliferation after 28 passages, were used at passages 4–6 and 1–2, respectively. Nuclear transfer and embryo culture procedures were essentially as described previously [Shi et al., 2003 Biol. Reprod. 69, 301–309]. Data were compared using chi-square test; differences were considered significant for P < 0.05. There were no significant differences in the fusion and cleavage rates between all three groups [BFF-GFP-Tag: 233/248 (94%) and 167/233 (72%); BFF-GFP: 274/294 (93%) and 216/274 (79%); BFF: 129/136 (96%) and 97/129 (75%)]. However, development to the blastocyst stage was significantly lower (P < 0.05) in the BFF-GFP-Tag group [5/233 (2%)] than in the BFF-GFP and BFF groups [110/275 (40%) and 53/129 (41%), respectively)]. From our results we conclude that SV40Tag-transduced bovine fetal fibroblasts with high proliferative potential may have lost the ability to respond to the normal cell cycle controls and, as a consequence, have low developmental potential for nuclear transfer.
