DESIGN AND CONSTRUCTION OF HUMAN ARTIFICIAL CHROMOSOME VECTORS

人类人工染色体载体的设计和构建

• 批准号: 2576538
• 负责人: P P LIU
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2576538
• 关键词: biotechnology; chromosomes; fusion gene; gene expression; gene targeting; genetic library; genetic manipulation; genetic markers; nucleic acid sequence; regulatory gene; technology /technique development; tissue /cell culture; transfection /expression vector

Small, truncated chromosomes have been associated with certain diseases and occasionally identified in the normal populations, as well as generated experimentally. These mitotically stable mini-chromosomes are very valuable for the identification and characterization of functional chromosome elements and could be converted to manipulable human artificial chromosomes (HAC). We acquired four cell lines containing mini-chromosomes, three of them from normal individuals and one generated experimentally. The mini-chromosomes range from 3 to 10 mb in size and their chromosomal origins were determined. Microdissection of the mini-chromosomes was performed, and the microdissected DNA was used to screen chromosome-specific cosmid libraries. These studies have revealed that these mini-chromosomes contain limited euchromatic sequences and truncated centromeres. Further analysis at the sequence level is in progress which may identify functional subfamilies of the repeated satellite DNA at human centromeres. Additional physical mapping analysis will help to define other necessary sequences for chromosome functions. Selectable markers will be inserted into the mini-chromosomes through homologous recombination and the feasibility of using these mini-chromosomes as HAC vectors will be tested.

截短的小染色体与某些疾病有关 偶尔在正常人群中发现， 实验产生的。这些有丝分裂稳定的微型染色体 对于功能性的鉴定和表征非常有价值， 染色体元件，并可以转换为可操纵的人类 人工染色体（HAC）。我们获得了四种细胞系， 微型染色体，其中三个来自正常个体，一个来自 实验性的微型染色体的大小从3到10 mb不等， 确定了它们的染色体来源。显微解剖 进行了微型染色体，并将显微切割的DNA用于 筛选染色体特异性粘粒文库。这些研究揭示 这些微型染色体含有有限的常染色质序列， 截短的着丝粒。在序列水平上的进一步分析在 这一进展可能会确定功能亚家族的重复 人类着丝粒的卫星DNA其他物理映射分析 将有助于确定染色体功能的其他必要序列。 可选择的标记将被插入到微型染色体中， 同源重组和使用这些的可行性 将测试作为HAC载体的微型染色体。