利用半克隆技术筛选参与早期胚胎发育的关键雌性印记基因

Maternally imprinted genes are paternally expressed genes that play critical roles during embryonic development. Recent high-throughput analyses result in many potential maternally imprinted genes that exhibit paternal expression bias. These candidates need to be further confirmed. Meanwhile, their functions in embryonic development should be comprehensively investigated. However, regular genome-editing methods may not be possible to quickly identify important genes involved in embryonic development from these potential candidates. Recently, we established semi-cloned technology, in which, androgenetic haploid embryonic stem cells (AG-haESCs) could be used in place of sperm to efficiently produce health semi-cloned (SC) mice. We then proposed that, if the maternally imprinted gene were essential for embryonic development, deletion of the gene in AG-haESCs would result in SC embryos without its expression during the whole process of embryonic development, highly likely leading to abnormal embryonic development. In this study, we will firstly test whether SC technology is feasible for quick screening of maternally imprinted genes by mutation of Peg10, a well-known maternally imprinted gene whose paternal deletion leads to embryonic lethal at 10.5 days post-coitum, in AG-haESCs, and followed by injection of mutant cells into oocytes to observe the phenotype of embryonic development at different time. If the SC embryos show early embryonic lethality owing to defects in the placenta that is same as the phenotype of Peg10 knockout mice, we will then use this system to screen important maternally imprinted gene from 14 selected genes that exhibit paternal expression bias. We will establish AG-haESCs carrying mutant genes and by injection of them into oocytes, produce SC embryos to analyze the phenotypes of embryonic development. We will choose 1 or 2 essential genes for embryonic development for further functional analyses and revealing the underlying mechanisms. We believe that, from our proposed study, we will not only establish a new system for analysis of imprinted genes, but also reveal some essential imprinted genes for embryonic development.

雌性印记基因是一类母本不表达而父本表达的基因，在胚胎发育中起重要作用。高通量测序研究揭示了一批极度父本表达的基因（候选雌性印记基因），亟待进一步验证和功能分析。然而，传统的基因编辑方法难以快速确认哪些雌性印记基因在胚胎发育中起重要作用。最近，本实验室建立将培养的孤雄单倍体胚胎干细胞注入卵子中高效获得健康的半克隆小鼠的半克隆技术。我们推测，如果候选雌性印记基因在胚胎发育中起重要作用，父本（孤雄单倍体干细胞）基因的敲除会导致该基因表达在胚胎发育过程中完全缺失，从而出现严重的发育异常。基于此，本研究先以已知在胚胎发育中起重要作用的雌性印记基因Peg10作为对象验证半克隆技术用于开展此类研究的可行性。进而选择14个报道的雌性候选印记基因，在单倍体细胞中逐一敲除并建立细胞系，通过卵子注射获得半克隆胚胎，选择有严重发育表型的基因进行深入研究。本研究将建立一个胚胎发育表观遗传调控研究的新体系。

印记基因是一类单一亲本表达的基因，在胚胎发育中发挥着重要的作用。然而，由于缺乏高效的研究手段，发育中的印记调控研究受到很大的限制。我们通过核移植的方法将精子重编程为单倍体的胚胎干细胞（又称为类精子干细胞），并证明其能替代精子通过卵子注射产生半克隆小鼠（半克隆技术）。进一步研究揭示在类精子干细胞中敲除两个雄性印记基因调控区域，可以将半克隆小鼠的发育效率提高10倍。这一结果提示半克隆技术可能是研究印记基因调控发育的重要手段。本项目希望通过在类精子干细胞中敲除可能的雌性印记基因，并将其注入卵子产生胚胎，研究这些基因是否会对胚胎发育的影响；开展基于印记调控的单亲发育研究；拓展并进一步优化半克隆技术；开展基于半克隆的复杂基因编辑小鼠的构建。在本项目的支持下，我们取得了以下重要进展。1，在发育的印记调控研究中，实现基于半克隆技术的雌性印记基因的筛选，发现四个可能的参与胚胎发育的雌性印记基因进行深入研究；通过印记调控使得孤雄单亲胚胎发育延长到出生后23天；揭示重要的雄性印记调控区对胚胎发育的时序调节；发现雄性印记的表观完整性增强类精子干细胞的发育潜能。2，在半克隆技术的拓展和优化研究中，建立人的类精子干细胞；建立过滤富集单倍体胚胎干细胞的方法。3，在半克隆技术的应用研究中，建立基于半克隆技术的关键氨基酸的在体遗传筛选体系；建立基于半克隆技术的骨发育中关键基因的在体遗传筛选体系；实现基于半克隆技术构建染色体融合小鼠模型模拟染色体演化过程；开展基于半克隆技术的基因组标签计划。在本项目的支持下，我们还应邀为PLOS Genetics发表关于基因印记的研究手段的综述；应邀为Biology of Reproduction创刊50周年撰写半克隆技术应用的综述；应邀为NSR组织基因编辑专刊并撰写Perspective介绍基因组标签计划。本项目研究证明半克隆技术是研究发育的印记调控的有效手段，为进一步揭示复杂的调控机制奠定了基础。