ANALYSIS OF HUMAN ARTIFICIAL CHROMOSOME CONSTRUCTS IN MAMMALIAN CELLS

哺乳动物细胞中人类人工染色体结构的分析

• 批准号: 6162597
• 负责人: M ROSENFELD
• 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/6162597
• 关键词: DNA replication origin; artificial chromosomes; clone cells; genetic mapping; genetic techniques; human genetic material tag; molecular cloning; telomere; transfection /expression vector

We have constructed human artificial chromosomes (HACs) by modifying two different yeast artificial chromosomes (YACs) containing human centromeric a satellite DNA with human telomere sequences and putative origins of replication. The resulting HACs (250 and 1000 kb) which contain a neomycin resistance gene (neo r)have been introduced into mammalian cell lines and analyzed for their chromosomal function. Initial studies using fluorescence in situ hybridization (FISH) indicated that the HACs delivered to CHO cells by spheroplast fusion were integrated into the CHO genome. However, centromeric function was suggested for the larger HAC by the presence of a constriction and centromere binding proteins at the site of integration, and by the association of this integrated HAC with the induction of aneuploidy over time. Subsequently, both HACs have been transferred to human cells by liposome-mediated transfection of gel-purified DNA. Extrachromosomal elements containing the introduced a satellite DNA have been identified in neor transfectants generated from each HAC in the absence of integration into the human genome. Preliminary stability assessment indicates that the 1000 kb HAC is stable in the absence of G418 selection through 45 generations. We are currently pursuing these stability studies further. In addition, we are assessing the integrity and structural nature of the HACs compared to the material which we originally introduced into the human cells. Projects underway include (1) the introduction of the HAC into ES cells to generate transgenic mice; (2) developing strategies to transfer the HAC back to yeast for propagation, analysis and comparison to the original HAC; and (3) developing strategies to incorporate potentially therapeutic genes into a functional HAC.

我们通过修改两个构建了人造染色体（HAC） 包含人类的不同酵母人工染色体（YAC） CencerRomeric A卫星DNA具有人类端粒序列和推定的卫星DNA 复制的起源。由此产生的HAC（250和1000 KB） 含有新霉素抗性基因（NEO R）已被引入 哺乳动物细胞系并分析其染色体功能。最初的 使用荧光原位杂交（FISH）的研究表明 整合了通过护理融合传递到CHO细胞的HACS 进入CHO基因组。但是，建议对中心粒功能 通过存在收缩和丝粒结合，更大的HAC 蛋白质在整合位点，并通过此关联 随着时间的流逝，HAC与非整倍性诱导。随后， 两种HAC已通过脂质体介导的人转移到人类细胞中 转染凝胶纯化的DNA。包含外染色体元素 引入的卫星DNA已在神经转染物中鉴定 在没有整合到人的情况下从每个HAC产生 基因组。初步稳定性评估表明1000 kb HAC 在没有G418至45代的G418选择的情况下是稳定的。我们是 目前正在进一步追求这些稳定研究。此外，我们是 评估HAC的完整性和结构性与 我们最初引入人类细胞的材料。项目 正在进行中包括（1）将HAC引入ES细胞中 产生转基因小鼠； （2）制定转移HAC的策略 返回酵母进行繁殖，分析和比较原始 HAC; （3）制定策略以纳入潜在的治疗性 基因成功能性HAC。