Construction and application of human artificial chromosome vectors for stable gene expression by minichromosome-modifying system.

为了了解人类人工微染色体中不同区域基因表达的位置效应和稳定性，我们构建了四种不同类型的修饰微染色体，其中引入了含有完整基因单元的BAC（HPRT，或因子IX）进入各种位点，如近端粒、近着丝粒和常染色质区，并且我们研究了基因表达的强度和稳定性。我们构建了质粒载体，通过同源重组或端粒靶向的方法将lox 71（突变loxP）导入人微小染色体的不同位点。我们构建了端粒靶向载体，将neoR基因替换为位于微染色体端粒区的ZeoR基因，并利用上述载体对微染色体进行修饰，通过Cre/mutant lox重组系统将BAC（HPRT-66或F9-66）导入到修饰后的微染色体中。在HPRT-66 BAC和F9-66 BAC中，BAC的导入效率分别为75%和70%，qPCR法测得HPRT基因在端粒、近着丝粒和常染色质区的相对表达量分别为100、83和122。DT 40细胞中修饰的微型染色体的拷贝数为1拷贝/核（约70%）或2拷贝/核（约30%）。人HPRT基因的相对表达量为DT 40中真实Hprt基因表达量的15%。在培养60天后，任何修饰的微型染色体中的基因表达没有显著变化。

In order to know the position effect and stability of gene expression at various regions in human artificial minichromosomes, we have constructed four types of modified minichromosomes, which was introduced a BAC harboring a whole gene unit (HPRT, or Factor IX) into various sites such as near telomere, near centromere, and euchromatic region, and we investigated intensity and stability of the gene expression.Fist, we constructed plasmid vectors, which could introduce lox71 (mutant loxp) into the human minichromosome at various sites by homologous recombination or telomere targeting method. We also constructed a telomere-targeting vector, which could replace the neoR gene to the ZeoR gene located at the telomeric region in the minichromosome.Then, we modified the minichromosome using the vectors above, and introduced a BAC (HPRT-66 or F9-66) into the modified minichromosomes by the Cre/mutant lox recombination system. The efficiencies of BAC introduction into the minichromoscmes were approximately 75% and 70% for HPRT-66 BAC and F9-66 BAC, respectively.Relative gene expressions of human HPRT gene estimated by qPCR were 100, 83, and 122, when the gene was located near telomere near centromere, and euchromatic region, respectively. Copy number of the modified minichromosomes in DT40 cells was 1 copy/nucleus (approximately 70%) or 2 copies/nucleus (approximately 30%). Relative expression of the human HPRT gene was 15% of authentic Hprt gene expression in DT40. The gene expression was not significantly changed in any modified minichromosomes after 60 days culture.