80DEVELOPMENTAL POTENTIAL OF BOVINE NUCLEAR TRANSFER EMBRYOS DERIVED FROM FETAL FIBROBLASTS TRANSDUCED WITH SIMIAN VIRUS 40 LARGE T ANTIGEN

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.