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Analysis and Selection for gene-amplified cell based on FISH

基于FISH的基因扩增细胞分析与选择

基本信息

批准号：
11650817
负责人：
OMASA Takeshi
金额：
$ 2.18万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11650817/
关键词：
gene-amplification FISH F-MTX flow cytometer FISH (Fluore scence in situ hybridization)dhtr FISH(Fluorescene in situ hybridization)FISH

项目摘要

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

为了建立一种简便、快速的构建方法，以获得稳定、高产的基因扩增重组中国仓鼠卵巢(CHO)细胞系，进行了各种甲氨蝶呤(MTX)的分步筛选。比较细胞的特定生长率和产量，并用荧光原位杂交(FISH)确定扩增基因位置的分布。在MTX浓度递增最缓慢的选择条件下，细胞池的比生长和产量达到最高水平，约82%的扩增基因位于端粒区附近。为了阐明基因扩增细胞池在逐步增加MTX浓度过程中的行为，我们研究了从基因扩增细胞池中分离出来的基因扩增克隆。结果，扩增的基因位于端粒区附近的端粒型克隆是…与其他类型的无性系相比，更多的无性系更稳定、更高产。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，许多其他类型的克隆的扩增基因不到10个拷贝。在没有甲氨蝶呤的长期培养过程中，在其他类型的克隆中，扩增的基因在染色体DNA中迅速减少。使用异硫氰酸荧光素标记的甲氨蝶呤(F-MTX)试剂和流式细胞术，我们能够很容易地区分高产细胞和其他类型的细胞。根据对MTX抗性的不同，荧光水平也不同。基于这一新发现，与有限稀释法相比，从异源基因扩增细胞库中分离出高产量的基因扩增细胞更容易。限制稀释法需要几个月的时间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的时间。较少