适用于人胚胎干细胞的大型合成染色体导入策略

Human genome project (HGP)-write following the HGP-read is a major research plan which aims to help us further understand the structure and function of human genome and promote the development of life science and medicine. Chromosome redesign, synthesis, assembly and transplantation are routine steps involved in this project, but the rules for chromosome/genome redesign are still being explored. Human embryonic stem cells (hESCs) are vital materials for embryonic development and in vitro regenerative medicine research. Introduction of synthetic chromosomes into hESCs for functional verification, especially using the differentiation potential of hESCs in vitro, can better establish the rules for synthetic chromosome design. Meanwhile, synthetic genomics can provide technique solutions for the mechanism research of stemness maintenance, differentiation, etc. in hESCs. Currently, it is infeasible to directly assemble synthetic chromosomes in hESCs. As an alternative, synthetic chromosomes is assembled in yeast and this platform has been well established in our laboratory. However, it is still a huge challenge to deliver large size synthetic chromosomes into hESCs. This project aims to develop a bi-functional surface displaying reporter system for transgenic hESCs labeling and sorting which would be used in the assessment of synthetic chromosome delivery strategies. We are going to reveal the DNA size limit of currently available DNA delivery methods, including liposome or cationic polymer-mediated transfection and electrotransfection, for synthetic chromosome delivery in hESCs and optimize these methods to be competent for the transplantation of synthetic chromosome in size of 200-600 kb. We will also test the feasibility of yeast spheroplast fusion method for synthetic chromosome tansplantation in hESCs and optimize the microcell mediated chromosome transfer strategy, which aims to provide solutions for the transplantation of synthetic chromosome over 600 kb. And eventually, a technical system for large synthetic chromosome transplantation would be established and contribute to accelerating the implementation and application of HGP-write.

人类基因组编写计划涉及大型染色体的设计、合成、组装及移植。人胚胎干细胞（hESCs）是体外研究胚胎发育及再生医学的重要材料，将合成染色体导入hESCs，通过干性维持及诱导分化验证其功能，有望更好的探索合成染色体的设计原则；同时也可利用染色体重设计与合成的手段研究hESCs的干性维持、分化等机制。实验室前期建立了完善的合成染色体组装技术体系但后续合成染色体植入hESCs尚无有效的技术手段。本项目拟建立用于hESCs标记及分选的表面展示型报告系统，辅助合成染色体移植方法的适用性评估；对脂质体法等化学法及电转法进行测试及优化，为200-600 kb的合成染色体植入hESCs提供解决方案；探讨酵母原生质体融合在hESCs上的适用性，同时对微细胞介导法进行优化，为600 kb以上载体的递送提供解决方案；最终建立高效的hESCs合成染色体植入技术体系，加速人类基因组编写计划的实施及应用。

人类基因组编写计划涉及大型染色体的设计、合成、组装及移植。人胚胎干细胞（hESCs）是体外研究胚胎发育及再生医学的重要材料，将合成染色体导入hESCs，通过干性维持及诱导分化验证其功能，有望更好的探索合成染色体的设计原则；同时也可利用染色体重设计与合成的手段研究hESCs的干性维持、分化等机制。实验室前期建立了完善的合成染色体组装技术体系但后续合成染色体植入hESCs尚无有效的技术手段。. 本研究旨在探索常规化学转染法，酵母原生质体融合法，微细胞介导的染色体转移法（MMCT）用于hESCs大型DNA载体导入的可行性；建立hESCs的常规转染方法及基因定点敲入技术，设计多重组位点的landing pad，构建携带重组位点的稳定细胞株；开发基于CRISPR/Cas9的通用的DNA载体定点敲入策略，最终为实现大型DNA载体导入hESCs并实现稳定遗传提供解决方案。. 本研究尝试了基于阳离子聚合物的化学转染法，可在hESCs中进行150 kb的模型载体的转染，但是未能筛选获得稳定细胞株，实现大型DNA载体的高效定点整合是有效的代偿方案。设计了一种基于CRISPR/Cas9通用性DNA载体定点敲入策略，并在293T、HT1080细胞中进行了常规载体及大型载体的敲入测试；建立了hESCs的常规转染及基于CRISPR/Cas9的外源基因定点敲入技术，构建了携带loxP-Neo landing pad的hESCs细胞株。设计构建了包含多种特异性重组位点的landing pad并将其定点敲入至293T细胞的AAVS1位点获得稳定细胞株。以上结果将为后续深入开发hESCs的合成染色体导入技术奠定基础。

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