72 EFFECT OF CELL CYCLE PHASE OF GENE-MANIPULATED FETAL FIBROBLASTS ON THE DEVELOPMENT OF CLONED BOVINE EMBRYOS

Abstract

Transgenic bovine fetuses and offspring can be produced by using gene-modified somatic cells and clones of these cells. In this study, we examined the effects of specific cell cycle (early G1 phase) of donor cell (gene-manipulated fibroblasts) on the development of the nuclear transfer (NT) embryos into blastocysts and on the fetus production after embryo transfer. The gene-manipulated (tg; targeting of one or both alleles of gene encoding α-1,3-galactosyltransferase) or non-manipulated (control) bovine fetal fibroblasts were used for NT. The fibroblasts transfected with the targeting vector were selected with 0.4 mg mL⁻¹ G418. The G418-resistant cells were monitored by PCR and Southern blot analysis. The cells (tg cells) in which homologous recombination occurred were used for NT. For NT, both tg cells and control cells were cultured in DMEM with 10% FCS. Early G1 cells were prepared by choosing pairs of bridged cells derived from mitotic phase cells (Urakawa M et al. 2004 Theriogenology 62, 714–728), and non-synchronized cells were obtained from a culture plate that had reached 60–80% confluence. Each donor cell was inserted into an enucleated, in vitro-matured (19 h) oocyte. Oocyte-cell couples were electrofused and activated with calcium ionophore and cycloheximide. The NT embryos were then co-cultured with bovine oviduct epithelial cells in CR1aa with 5% CS. The blastocyst rates were determined at 6 days after NT. The blastocysts were nonsurgically transferred to recipient heifers, and the developmental rate to the normal fetus was examined by the recovery of fetus or by using ultrasonography at Days 35–42. Data were analyzed by ANOVA. The developmental rate to the blastocyst stage did not differ significantly between tg (28.4%, 128/425) and control (25.4%, 181/739) cell groups. In the control group, the blastocyst rate of embryos constructed from early G1 phase fibroblasts (25.7%, 80/311) was not significantly different from that of embryos constructed with non-synchronized fibroblasts (23.6%, 101/428). In contrast, the blastocyst rate of tg cell derived-embryos was lower (P < 0.05) in early G1 phase (23.5%, 71/302) than in non-synchronized cell phase (46.3%, 57/123). The rate of development to a normal fetus in the tg group (15.4%, 4/26) was significant lower than that in the control group (62.5%, 25/40). For both the tg group and the control group, the rate of development to fetus tended to be higher (P > 0.05) for blastocysts derived from cells at the early G1 phase than for blastocysts derived from non-synchronized cells (tg group, 25.0%, 3/12 v. 7.1%, 1/14; control group, 90.0%, 9/10 v. 53.3%, 16/30). These results demonstrate that gene modification of fetal fibroblasts affects the development of NT embryos to fetuses. In addition, the synchronization of genetically modified donor cells to the early G1 phase may increase the potential to develop to a normal fetus.