Laboratories of Molecular Pharmacology, National Cancer Institute, Bethesda, MD 20892, USA.
Nucleic Acids Res. 2013 May 1;41(10):e107. doi: 10.1093/nar/gkt205. Epub 2013 Apr 4.
Human artificial chromosome (HAC)-based vectors represent an alternative technology for gene delivery and expression with a potential to overcome the problems caused by the use of viral-based vectors. The recently developed alphoid(tetO)-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of tTS chromatin modifiers to its centromeric tetO sequences. This provides unique control for phenotypes induced by genes loaded into the alphoid(tetO)-HAC. However, inactivation of the HAC kinetochore requires transfection of cells by a retrovirus vector, a step that is potentially mutagenic. Here, we describe an approach to re-engineering the alphoid(tetO)-HAC that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. In the new HAC vector, a tTS-EYFP cassette is inserted into a gene-loading site along with a gene of interest. Expression of the tTS generates a self-regulating fluctuating heterochromatin on the alphoid(tetO)-HAC that induces fast silencing of the genes on the HAC without significant effects on HAC segregation. This silencing of the HAC-encoded genes can be readily recovered by adding doxycycline. The newly modified alphoid(tetO)-HAC-based system has multiple applications in gene function studies.
基于人类人工染色体（HAC）的载体代表了一种基因传递和表达的替代技术，具有克服基于病毒载体使用所带来的问题的潜力。最近开发的α-卫星（tetO）-HAC 优于其他 HAC 载体，因为通过 tTS 染色质修饰物与其着丝粒 tetO 序列的结合，使 HAC 动粒失活，从而可以轻易地从细胞中消除它。这为装入α-卫星（tetO）-HAC 的基因所诱导的表型提供了独特的控制。然而，HAC 动粒的失活需要通过逆转录病毒载体转染细胞，这一步骤具有潜在的诱变作用。在这里，我们描述了一种对α-卫星（tetO）-HAC 进行工程改造的方法，该方法允许在不进行转染步骤的情况下，验证归因于 HAC 中基因表达的表型变化。在新的 HAC 载体中，tTS-EYFP 盒与感兴趣的基因一起插入基因加载位点。tTS 的表达在α-卫星（tetO）-HAC 上产生了一种自我调节的波动异染色质，可快速沉默 HAC 上的基因，而对 HAC 分离没有显著影响。通过添加多西环素可以很容易地恢复 HAC 编码基因的沉默。新修饰的基于α-卫星（tetO）-HAC 的系统在基因功能研究中有多种应用。
