Lethality, embryos from the Ggn+/2 intercrosses were harvested for genotyping at various stages of gestation between days 7.5?3.5 post-coitum (E7.5 13.5). No Ggn2/2 embryos were observed at or beyond E7.5 (Table 1). These data, and the absence of any evidence of embryo resorption, suggested that Ggn2/2 embryos died prior to implantation. Indeed, the genotyping of E2.5 3.5 embryos collected fromGGN Regulates Embryogenesis and Meiotic DSB Repairtimed Ggn+/2 intercrosses revealed that only 2 of Ggn2/2 embryos survived to the morula stage (Table 1 and Figure 2C). An analysis of 2-cell embryos obtained by in vitro fertilisation (IVF) using oocytes and sperm from Ggn+/2 mice however showed that Ggn2/2 embryos were present in the expected Mendelian distribution (Table 1). Together, these results indicate that the majority of Ggn2/2 embryos survive the first mitotic division, but all 23727046 die prior to implantation. Thus, our data define the essential physiological role for GGN in the maintenance of embryo viability at the very earliest events of pre-implantation embryo development. Next, we Title Loaded From File sought to determine if GGN is required for ES cell viability. Cells carrying two sets of the neomycin resistance cassette are more resistant to G418 than are those with a single copy of the cassette [19]. The two independently targeted Ggn+/2 ES cells were subjected to clonal selection in the presence of a higher dose of G418 in attempts to generate Ggn2/2 ES cells by gene conversion. No Ggn2/2 ES cells were identified from 182 ES colonies examined, suggesting that GGN is required for the establishment of viable ES cells. The inability to isolate Ggn2/2 ES cells in concert with the absence of Ggn2/2 embryos beyond the morula stage strongly suggest that GGN is essential for cell viability.Ggn is Ubiquitously Expressed at Low Levels in Preimplantation Embryos and Somatic CellsIn addition to high levels of expression in the testis, low levels of Ggn transcripts were detected in ovulated oocytes and preimplantation embryos as determined by a quantitative RT-PCR (qRT-PCR) assay that detected Ggn1, Ggn2 and Ggn3 transcripts (Figure 2D). Further, we observed low levels of Ggn expression in the majority of adult mouse (10 weeks-old) tissues examined (Figure 2E). Thus Ggn is expressed more widely than previously anticipated, suggesting a role for GGN in the development of both germ and somatic cells. Indeed, this hypothesis is strongly supported by the embryonic O K7 (A, D) and K18 (B, E). Merged images (C Lethality of the Ggn2/2 embryos. The death of the vast majority of Ggn2/2 embryos prior to morula and an absence by the blastocyst stage, is consistent with 15900046 a loss of viability once maternal Ggn mRNA stores are depleted i.e. declined expression in morula (Figure 2D).Ggn+/2 Pachytene Spermatocytes Show an Increased Incidence of Unrepaired DSBsNext, we investigated whether GGN plays a role in DSB repair during male meiosis. However, this could not be explored on Ggn2/2 male mice because of embryonic lethality, so we asked instead if meiotic DSB repair would be impaired by GGNhaploinsufficiency in the Ggn+/2 mice. To confirm haploinsufficiency, we performed qRT-PCR and GGN1 immunoblotting on purified spermatocytes from the Ggn+/+ and Ggn+/2 mice. We showed that Ggn transcripts in the Ggn+/2 spermatocytes were reduced to about 60 of the Ggn+/+ spermatocytes (Figure 3A). Consistently, GGN1 immunoblotting showed a reduction of GGN1 protein compared to that of Ggn+/+ spermatocytes (Figure 3B). SPO11-induced DSBs occur.Lethality, embryos from the Ggn+/2 intercrosses were harvested for genotyping at various stages of gestation between days 7.5?3.5 post-coitum (E7.5 13.5). No Ggn2/2 embryos were observed at or beyond E7.5 (Table 1). These data, and the absence of any evidence of embryo resorption, suggested that Ggn2/2 embryos died prior to implantation. Indeed, the genotyping of E2.5 3.5 embryos collected fromGGN Regulates Embryogenesis and Meiotic DSB Repairtimed Ggn+/2 intercrosses revealed that only 2 of Ggn2/2 embryos survived to the morula stage (Table 1 and Figure 2C). An analysis of 2-cell embryos obtained by in vitro fertilisation (IVF) using oocytes and sperm from Ggn+/2 mice however showed that Ggn2/2 embryos were present in the expected Mendelian distribution (Table 1). Together, these results indicate that the majority of Ggn2/2 embryos survive the first mitotic division, but all 23727046 die prior to implantation. Thus, our data define the essential physiological role for GGN in the maintenance of embryo viability at the very earliest events of pre-implantation embryo development. Next, we sought to determine if GGN is required for ES cell viability. Cells carrying two sets of the neomycin resistance cassette are more resistant to G418 than are those with a single copy of the cassette [19]. The two independently targeted Ggn+/2 ES cells were subjected to clonal selection in the presence of a higher dose of G418 in attempts to generate Ggn2/2 ES cells by gene conversion. No Ggn2/2 ES cells were identified from 182 ES colonies examined, suggesting that GGN is required for the establishment of viable ES cells. The inability to isolate Ggn2/2 ES cells in concert with the absence of Ggn2/2 embryos beyond the morula stage strongly suggest that GGN is essential for cell viability.Ggn is Ubiquitously Expressed at Low Levels in Preimplantation Embryos and Somatic CellsIn addition to high levels of expression in the testis, low levels of Ggn transcripts were detected in ovulated oocytes and preimplantation embryos as determined by a quantitative RT-PCR (qRT-PCR) assay that detected Ggn1, Ggn2 and Ggn3 transcripts (Figure 2D). Further, we observed low levels of Ggn expression in the majority of adult mouse (10 weeks-old) tissues examined (Figure 2E). Thus Ggn is expressed more widely than previously anticipated, suggesting a role for GGN in the development of both germ and somatic cells. Indeed, this hypothesis is strongly supported by the embryonic lethality of the Ggn2/2 embryos. The death of the vast majority of Ggn2/2 embryos prior to morula and an absence by the blastocyst stage, is consistent with 15900046 a loss of viability once maternal Ggn mRNA stores are depleted i.e. declined expression in morula (Figure 2D).Ggn+/2 Pachytene Spermatocytes Show an Increased Incidence of Unrepaired DSBsNext, we investigated whether GGN plays a role in DSB repair during male meiosis. However, this could not be explored on Ggn2/2 male mice because of embryonic lethality, so we asked instead if meiotic DSB repair would be impaired by GGNhaploinsufficiency in the Ggn+/2 mice. To confirm haploinsufficiency, we performed qRT-PCR and GGN1 immunoblotting on purified spermatocytes from the Ggn+/+ and Ggn+/2 mice. We showed that Ggn transcripts in the Ggn+/2 spermatocytes were reduced to about 60 of the Ggn+/+ spermatocytes (Figure 3A). Consistently, GGN1 immunoblotting showed a reduction of GGN1 protein compared to that of Ggn+/+ spermatocytes (Figure 3B). SPO11-induced DSBs occur.