Organization of Synthetic Alphoid DNA Array in Human Artificial Chromosome (HAC) with a Conditional Centromere
Organization of Synthetic Alphoid DNA Array in Human Artificial Chromosome (HAC) with a Conditional Centromere
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DOI：
10.1021/sb3000436
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发表时间：
2012-12-01
影响因子：
4.7
通讯作者：
Larionov, Vladimir
中科院分区：
文献类型：
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作者：
Kouprina, Natalay;Samoshkin, Alexander;Larionov, Vladimir
人类人工染色体（HACs）代表了一种新的有前途的附加型系统的功能基因组学，基因治疗和合成生物学。在转染到人细胞中时，从天然和合成的α DNA阵列工程化HAC。使用HAC进行基因表达研究需要了解其结构组织。然而，迄今为止构建的从头HAC都没有详细的物理映射。最近，我们构建了一种合成的alphoid（tetO）-HAC，它成功用于表达全长基因，以纠正人类细胞的遗传缺陷。HAC可以通过其条件动粒的失活而容易地从细胞群体中消除。这种独特的功能提供了一个控制表型变化归因于表达的HAC-encoded基因。这项工作描述了在alphoid（tetO）-HAC中的兆碱基大小的合成alphoid DNA阵列的组织，所述alphoid（tetO）-HAC已经由类似于50 kb的合成alphoid（tetO）-构建体形成。我们的分析表明，这个阵列代表了一个1.1 Mb的连续序列组装从多个拷贝的输入DNA，其中很大一部分是重新排列组装前。HAC中的串联和反向α型DNA重复序列的大小范围为25至150 kb。此外，我们证明了HAC的结构和功能结构域在几轮转移到不同的宿主细胞后保持不变。alphoidt（tetO）-HAC结构的知识提供了在不同操作期间控制HAC完整性的工具。我们的研究结果也揭示了在人类细胞中从头MAC形成的机制。
Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy, and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of the de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoid(tetO)-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoid(tetO)-HAC that has been formed from a similar to 50 kb synthetic alphoid(tetO)-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoidt(tetO)-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo MAC formation in human cells.