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: 6290276
• 负责人: PU PAUL 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/6290276
• 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

In collaboration with Dr. Ashlock, we have constructed a human artificial chromosome (HAC) vector from a 1 megabase yeast artificial chromosome (YAC). The YAC contained an alpha satellite DNA insert and was modified to have human telomeres and a putative human replication origin. The HAC vector was introduced into human cells in culture and HACs were identified which were mitotically stable in the absence of selection for more than 100 generations. The HACs were not derived from telomere fragmentation of endogenous chromosomes, but were larger than the original vector, suggesting the possibility of amplification and rearrangement. We are currently in the process of improving the design and maintenance of HAC vectors, using yeast genetic manipulation technologies. The yeast strains with humanized telomerase template, recently generated in our lab, will be used for the propagation of new HAC vectors. In addition, we are using the transformation-associated recombination (TAR) cloning technique in the yeast to clone large, unrearranged pieces of human chromosome centromeres. These new approaches and improved features will hopefully result in the production of more stable and reproducible HACs. - biotechnology research, gene therapy, genetics, human genome research,

与Ashlock博士合作，我们从1兆碱基的酵母人工染色体（YAC）构建了人类人工染色体（HAC）载体。YAC含有α卫星DNA插入物，并被修饰为具有人类端粒和推定的人类复制起点。将HAC载体引入培养的人细胞中，并鉴定在不存在选择的情况下有丝分裂稳定超过100代的HAC。HACs不是来源于内源染色体的端粒片段化，而是大于原始载体，表明扩增和重排的可能性。我们目前正在使用酵母遗传操作技术改进HAC载体的设计和维护。本实验室新近获得的具有人源化端粒酶模板的酵母菌株将用于新的HAC载体的增殖。此外，我们正在酵母中使用转化相关重组（TAR）克隆技术来克隆大的、未重排的人类染色体着丝粒片段。这些新的方法和改进的功能将有望产生更稳定和可重复的HAC。- 生物技术研究，基因治疗，遗传学，人类基因组研究，