Analysis and Selection for gene-amplified cell based on FISH

为了建立一种简便、快速、稳定、高产的基因扩增重组中国仓鼠卵巢（CHO）细胞系构建方法，对多种甲氨蝶呤（MTX）进行了逐步筛选。比较细胞的特定生长率和产生率，并利用荧光原位杂交（fluorescence in situ hybridization， FISH）确定扩增基因位置的分布。在MTX浓度逐步增加最缓慢的选择条件下，细胞池的比生长率和产率达到最高水平；约82%的扩增基因位于端粒区附近。为了阐明MTX浓度逐步增加时基因扩增细胞池的行为，我们研究了来自基因扩增细胞池的分离基因扩增克隆。结果发现，扩增基因位于端粒区附近的端粒型克隆比其他类型的克隆更稳定，产量更高。端粒型克隆在染色体DNA中有超过100个拷贝的扩增基因，相比之下，大量其他类型的克隆的扩增基因少于10个拷贝。在缺乏MTX的长期培养过程中，在其他类型的克隆中，染色体DNA中扩增的基因迅速减少。使用荧光素异硫氰酸标记的甲氨蝶呤（F-MTX）试剂与流式细胞术，我们很容易区分高产细胞和其他类型的细胞。荧光水平根据对甲氨蝶呤抗性的不同而不同。基于这一新发现，从异种基因扩增细胞池中分离高产基因扩增细胞比用极限稀释法更容易。限制稀释法需要几个月才能获得高产的基因扩增细胞，而我们基于流式细胞术的选择方法只需要几周。少

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, various kinds of stepwise methotrexate (MTX) selection were carried out. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth and production rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82 % of amplified genes were observed near the telomeric region. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, we investigated isolated gene-amplified clones derived from gene amplified cell pools. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were … More found to be more stable and productive the other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA In contrast a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.Using a fluorescein isothiocyanatelabeled methotrexate (F-MTX) reagent with flow cytomery, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene amplified cell pools more easily than by the method of limiting dilution assay. The limiting-dilution method requires several months to obtain highly productive gene amplified cells, while our flowcytometry-based method of selection requires only a few weeks. Less