Omasa Takeshi, Cao Yihua, Park Joon Young, Takagi Yasuhiro, Kimura Shuichi, Yano Hidenori, Honda Kohsuke, Asakawa Shuichi, Shimizu Nobuyoshi, Ohtake Hisao
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Biotechnol Bioeng. 2009 Dec 1;104(5):986-94. doi: 10.1002/bit.22463.
Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene-amplified CHO DR1000L-4N cell line for genome-wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC-FISH). Thirteen BAC-FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR-deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165-kb DNA region containing exogenous Dhfr was cloned from the BAC library using high-density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome.
中国仓鼠卵巢（CHO）细胞系广泛应用于科学研究和生物技术领域。为了对CHO细胞系进行全基因组分析，从一个小鼠二氢叶酸还原酶（DHFR）基因扩增的CHO DR1000L - 4N细胞系构建了一个CHO基因组细菌人工染色体（BAC）文库。该CHO BAC文库由122,281个克隆组成，预计可覆盖整个CHO基因组5倍。通过使用BAC克隆作为杂交探针的荧光原位杂交（FISH）成像构建了CHO染色体图谱（BAC - FISH）。由于该细胞系的非整倍性，在一个DHFR缺陷的CHO DG44细胞系中，需要13个BAC - FISH标记克隆来鉴定所有20条单条染色体。为了确定外源Dhfr扩增子的基因组结构，使用高密度复制（HDR）滤膜和Southern印迹分析从BAC文库中克隆了一个包含外源Dhfr的165 kb DNA区域。核苷酸序列分析揭示了一种新的基因组结构，其中包含Dhfr的载体序列被CHO基因组的长反向序列夹在中间。
