Construction and application of human artificial chromosome vectors for gene introduction using minichromosomes.

我们已经建立了一个有效的系统，用于将包括完整基因单位的整个BAC克隆导入到基于人类X染色体的微小染色体上。利用带有Cre重组酶的突变loxP位点和对改造事件的直接选择，可以获得高效率的正确改造克隆。在大约75%的分析克隆中，含有完整的人HPRT基因的改良的BAC被正确地改造到DT40细胞的微染色体上。使用以前报道的方法对BAC进行改造(Kaname和Huxley，BioTechniques 31：273(2001))。对另一个含有人凝血因子IX基因的BAC克隆，重组率约为80%。对于含有neR表达单位的4.5kb的质粒，重组率为100%。在这两个克隆中，HPRT基因在微染色体中的表达水平大致相同。该系统可以将任何可克隆为BAC的基因或基因簇引入微染色体中进行基因传递，并尝试通过微细胞融合的方法将修饰后的微染色体转移到其他细胞(HT1080，HPRT(-))中。通过对G418抗性克隆的筛选，可以有效地分离跨染色体细胞。抗性克隆中，每细胞微染色体拷贝数在1~8个之间。然而，它的表达是高效和稳定的。因此，微染色体可作为基因治疗的染色体载体。

We have established an efficient system for introducing a whole BAC clone including an intact gene unit onto a minichromosome based on the human X chromosome. The use of mutant loxP sites with Cre recombinase and direct selection for the retrofitting event led to a high efficiency of correctly retrofitted clones. A modified BAC containing the intact human HPRT gene was correctly retrofitted onto the minichromosome in DT40 cells in approximately 75% of analysed clones. Modification of BACs for retrofitting was used method previously reported (Kaname and Huxley, BioTechniques 31:273(2001)). For another BAC clone containing human factor IX gene, the efficiency of retrofitting was about 80%. For a plasmid (4.5kb) having a neoR expression unit, the retrofitting efficiency was 100%. The HPRT gene is expressed from the minichromosome at approximately the same level in both of these clones. This system allows one to introduce any gene or gene cluster which can be cloned as a BAC into the minichromosome for gene delivery.We also tried to transfer the modified minichromosomes into other cells (HT1080,HPRT(-)) by microcell-fusion method. It was efficient to isolate the transchromosomic cells by screening of G418 resistant clones. Of the resistant clones, copy number of the minichromosome per cell was in range of 1-8. Its expression was, however, efficient and stable. Thus, the minichromosome would be used as a chromosome vector for gene therapy.