72 EFFECTS OF GENE EXPRESSION IN BOVINE EMBRYOS RECONSTRUCTED WITH FIBROBLASTS TRANSFECTED WITH LUCIFERASE GENE ON THE SUBSEQUENT DEVELOPMENT

Abstract

During embryo development, embryonic gene activation (EGA) is one of the first critical events. Inappropriate EGA results in failure of further development. We have reported that gene expression in bovine embryos reconstructed with fibroblasts begins at 48 hours postfusion (hpf) and reaches a maximum level at 60 hpf as detected by their bioluminescence following injection of chicken β-actin/firefly luciferase fusion gene (β-act/luc+) into their nuclei (Saeki et al., 2001 Theriogenology 55, 289). In the present study, effects of gene expression in embryos reconstructed with bovine fibroblasts transfected with luciferase gene on their subsequent development to the blastocyst stage were examined. Cultured bovine fibroblasts taken from an ear of a female calf were transfected with plasmid containing β-act/luc+/IRES/EGFP and neo^r using GeneJammer (StrataGene, La Jolla, CA, USA). Neomycin-resistant cells were selected by culturing with G418. Then, EGFP-positive colonies were further selected under fluorescence microscopy to obtain stably transfected cells. The transfected cells were cultured for several passages. Growing (50 to 60% confluence, GCs) and serum-starved cells (SCs) were used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20 h post maturation. Enucleated oocytes were electrofused with the cells, and activated with calcium ionophore and cycloheximide. Luminescence in the embryos was detected with an imaging photon counter at 0 and 60 hpf. Luminescence-positive (P) and -negative (N) embryos were cultured separately at each detection time. Embryos were cultured until 168 hpf, and examined for cleavage and blastocyst development. Experiments were repeated 3 times, and totals of 91 and 123 embryos were reconstructed with GCs and SCs, respectively. Data were analyzed with Fisher’s PLSD test following ANOVA by Stat View software (Ver. 5.0). At 0 hpf, luminescence was detected in 55 and 4% of embryos reconstructed with GCs and SCs, respectively. At 60 hpf, luminescence was detected in 47 and 28% of P and N embryos with GCs, and 17 and 40% of P and N embryos with SCs at 0 hpf, respectively. Cleavage rates were not different among groups (P > 0.05). Blastocysts were obtained only from the groups of embryos that were N at 0 hpf and P at 60 hpf (8% with GCs and 17% with SCs). No embryos in the other groups developed to the blastocyst stage. These results suggest that appropriate gene expression in embryos reconstructed with somatic cells is important for their subsequent development and that detecting the reporter gene expression can be used for selection of viable cloned embryos.