Role of OCT4 during preimplantation development of bovine embryos

OCT4在牛胚胎着床前发育过程中的作用

• 批准号: 405453332
• 负责人: Dr. Kilian Simmet
• 金额: --
• 依托单位: Lehrstuhl für Molekulare Tierzucht und Biotechnologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405453332?language=en
• 关键词: Role; OCT4; during; preimplantation; development

Evidence in literature suggests substantial differences in preimplantation development of bovine embryos to the extensively studied mouse model. Differences include the interaction of OCT4 and CDX2 in the trophectoderm as well as the role of FGF/MAPK signaling during the second lineage differentiation of the hypoblast. Enhanced knowledge of the mechanisms and their regulation involved in the first lineage differentiations during preimplantation development in bovine, as a model complementary to mouse, would increase our overall understanding of conserved and species-specific mechanisms of early lineage specification and selective maintenance of pluripotency during mammalian preimplantation embryo development. With OCT4 being involved in both regulation of pluripotency and differentiation, deciphering its function in bovine is of special interest. Therefore, using adult fibroblasts with a CRISPR/Cas9 induced knockout of OCT4 and somatic cell nuclear transfer (SCNT), we aim to elucidate the role of OCT4 during bovine preimplantation development. We specifically hypothesize, that the involvement of OCT4 during the second lineage differentiation differs between bovine embryos and published mechanisms in the mouse. Furthermore, we suggest that embryos from somatic cell nuclear transfer reflect the principle mechanisms of early lineage development observed in fertilized embryos and that loss of OCT4 in one of the two aggregation partners in embryonic chimeras induces bias in cell lineage allocation. Our work program will include immunofluorescence on markers specific to the cell lineages in the early embryo, i.e. GATA6 for hypoblast, NANOG for epiblast and CDX2 for trophectoderm, and transcriptome analysis by RNA sequencing. To confirm findings from embryos produced via SCNT, we want to produce OCT4 KO embryos through direct injection of CRISPR/Cas9 into in vitro fertilized zygotes. Additionally, we aim to elucidate the second lineage differentiation and the role of OCT4 during by transfer of embryos to recipient cows at day 7 and their recovery at day 12. This is to our knowledge the first study on the function of OCT4 during bovine preimplantation development using a reverse genetics approach and the first comprehensive investigation of the second lineage differentiation in bovine embryos.

文献中的证据表明，牛胚胎的着床前发育与广泛研究的小鼠模型存在实质性差异。差异包括OCT 4和CDX 2在滋养外胚层中的相互作用以及FGF/MAPK信号传导在下胚层第二谱系分化期间的作用。牛作为小鼠胚胎发育的补充模型，深入了解牛胚胎早期谱系分化的机制及其调控，将有助于我们全面了解哺乳动物胚胎早期谱系特化和多能性选择性维持的保守性和种属特异性机制。由于OCT 4参与多能性和分化的调节，因此破译其在牛中的功能具有特殊意义。因此，使用具有CRISPR/Cas9诱导的OCT 4敲除和体细胞核移植（SCNT）的成体成纤维细胞，我们的目标是阐明OCT 4在牛植入前发育期间的作用。我们特别假设，OCT 4在第二谱系分化过程中的参与在牛胚胎和小鼠中已发表的机制之间存在差异。此外，我们认为，体细胞核移植胚胎反映了受精胚胎中观察到的早期谱系发育的主要机制，胚胎嵌合体中两个聚集伙伴之一的OCT 4缺失诱导细胞谱系分配的偏倚。我们的工作计划将包括对早期胚胎细胞谱系特异性标记物的免疫荧光，即下胚层的GATA 6，上胚层的NANOG和滋养外胚层的CDX 2，以及通过RNA测序进行的转录组分析。为了证实通过SCNT产生的胚胎的发现，我们希望通过将CRISPR/Cas9直接注射到体外受精卵中来产生OCT 4 KO胚胎。此外，我们的目的是阐明第二谱系分化和OCT 4在胚胎移植到受体牛在第7天和他们的恢复在第12天的作用。据我们所知，这是第一次研究OCT 4在牛植入前发育过程中的功能，使用反向遗传学方法和第一次全面调查牛胚胎的第二谱系分化。