Abstract. In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken β-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4-to 8-cell stage at 60 hpf. Only 4-to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with wholeembryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4-to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.

During embryo development, embryonic gene activation (EGA) is the first critical event. We previously showed that EGA is also critical for further development in somatic cell-cloned embryos (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). To show this, we reconstructed bovine embryos with bovine somatic cells transfected with chicken β-actin/firefly luciferase fusion gene (β−act/luc+) and showed that only luminescent embryos at 60 hours post-fusion (hpf) developed to the blastocyst stage. In this study, we examined the relation between the intensity of expression of the same reporter gene in embryos reconstructed with bovine β−act/luc+ fibroblasts and their subsequent development to the blastocyst stage. Bovine fibroblasts were transfected with β−act/luc+ as described earlier (Saeki K et al. 2004 Reprod. Fertil. Dev. 16, 157–158 abst). The stably transfected and cloned cells were cultured for several passages. The cells were cultured under serum starvation (0.4% FCS) for 7 days and then 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 a calcium ionophore and cycloheximide. The LUC+ signal (luminescence) in the embryos was detected in medium containing 500 μg mL−1 luciferin with an imaging photon counter (ARGUS 50, Hamamatsu, Japan) for 30 consecutive min at 60 hpf. The intensity of luminescence in embryos (4- to 8-cell stage) was graded as being strong (>10 × 104 pixels/embryo), intermediate (5 to 10 × 104 pixels/embryo), weak (<5 × 104 pixels/embryo), or absent. The embryos were cultured separately until 168 hpf, and examined for blastocyst development. Experiments were repeated four times, and the data were analyzed with Fisher's PLSD test following ANOVA by Stat View software (Ver. 5.0; abacus Concepts, Berkeley, CA, USA). Of 125 embryos that were reconstructed, 74 (59%) developed to the 4- to 8-cell stage at 60 hpf. The luminescence was strong in 29 (39%) of the embryos, intermediate in 12 (16%), weak in 19 (26%), and absent in 14 (19%). Blastocysts were obtained from a group of embryos that exhibited strong luminescence (10/29, 34%), but none of the embryos from the other groups developed to blastocysts. These results suggest that active gene expression in embryos reconstructed with somatic cells is important for their subsequent development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Ministry of Education, Culture, Sports, Science, and Technology, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows for the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. However, the editing conditions and protocols to produce knockout lines have been optimised for a limited number of strains or stocks. Here, we demonstrate a novel and universal approach for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in 8 common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our novel approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research.

Recently, enhanced development to full term was obtained with embryos reconstructed with bovine early G1 cells rather than with G0 cells (Kasinathan et al. 2001 Nat. Biotechnol. 19, 1176-1178; Urakawa et al. 2004 Theriogenology 62, 714-728). However, the reason why donor somatic cells at the early G1 phase are better for embryo reconstruction is unclear. In this study, we investigated the relation of spatial gene expression patterns at the 4- to 8-cell stage to blastocyst development of embryos reconstructed with early G1 cells. Bovine fibroblasts stably transfected with �-act/luc+/IRES/EGFP were used for embryo reconstruction. M phase cells were prepared as described by Urakawa et al. (2004). Early G1 cells were obtained from cultured M phase cells soon after the M phase cells divided. Quiescent cells (cultured in 0.4% serum for 7 days) were used as G0 cells for a control. The cells were electrofused with enucleated bovine oocytes matured in vitro, and activated with a calcium ionophore and cycloheximide. The reconstructed embryos were cultured until 60 hours post fusion (hpf), and zonae pellucidae of 4- to 8-cell embryos were removed by pronase. To determine gene expression, the LUC+ activity (luminescence) in the embryo blastomeres was detected with an imaging photon counter (Hamamatsu Photonics, Hamamatsu City, Shikuoka Prefecture, Japan) for 10 min. The embryos were categorized as being positive, mosaic, or negative depending on whether all, some or no blastomeres were luminescent, respectively. The embryos were cultured in mSOF medium individually until 168 hpf to assess development to the blastocyst stage. Blastocyst development of reconstructed embryos without detection of luminescence was also examined. Experiments were repeated three times, and the data were analyzed with Fisher's PLSD test following ANOVA. At 60 hpf, 75% (74/99) of embryos reconstructed with early G1 cells and 55% (46/83) of embryos with G0 cells developed to 4- to 8-cell stage embryos. The difference is significant (P < 0.05). The percentages of positive, mosaic, and negative embryos with G1 cells were 49, 35 and 16%, and blastocyst rates were 30, 11, and 0%, respectively. With G0 cells, the percentages were 32, 56, and 12%, and the blastocyst rates were 15, 4, and 0%, respectively. More positive embryos were obtained with early G1 cells than with G0 cells (P < 0.05). Blastocyst rates of the positive embryos with early G1 cells were the same as with G0 cells (P > 0.05). Blastocyst development of positive embryos was higher than that of mosaic and negative embryos in early G1 and G0 groups (P < 0.05). Without detection of luminescence, the blastocyst rates from the reconstructed embryos were 43% (35/81) and 16% (20/125) with early G1 and G0 cells, respectively (P < 0.05). These results suggest that the higher developmental capacity of embryos reconstructed with early G1 cells might be related to the appropriate spatial gene expression at the 4- to 8-cell stage. A part of this study was supported by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.