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，我们通过1兆瓦酵母菌人工染色体（YAC）构建了人造染色体（HAC）载体。 YAC包含一个α卫星DNA插入物，并经过修改为具有人类端粒和推定的人类复制起源。将HAC载体引入了培养物中的人类细胞中，并鉴定出HACS，在没有选择的情况下，该HAC载体在有丝分裂的稳定性上是稳定的100代。 HACS并非来自内源性染色体的端粒破碎，而是大于原始载体，这表明可能会放大和重排。目前，我们正在使用酵母基因操纵技术来改善HAC载体的设计和维护。最近在我们的实验室中生成的具有人源化端粒酶模板的酵母菌菌株将用于传播新的HAC矢量。此外，我们正在使用酵母中与转化相关的重组（TAR）克隆技术来克隆大型的人类染色体centromeres的碎片。这些新方法和改进的功能希望能够产生更稳定和可重现的HAC。 - 生物技术研究，基因治疗，遗传学，人类基因组研究，