Introduction

Chinese hamster ovary (CHO) cells [1] are today a very important host for the commercial-scale production of protein pharmaceuticals. Two sub clones of CHO cells, proline-requiring CHO K1 [2] and the dihydrofolate reductase (DHFR) gene-deficient CHO DG44 [3], are the most widely used for both scientific research and industrial applications [10].

Results and discussion

Construction of BAC-based physical map for Chinese hamster ovary cells

A CHO genomic BAC library consisted of 122,281 clones was constructed in a previous study [7]. Three hundred BAC clones were randomly selected from this library and mapped onto each chromosome of CHO DG44 cells by BAC-FISH. The FISH signal location of BAC clone probes on each chromosome were determined by digital image analysis and expressed as FLpter values. The 185 BAC clones were also mapped on the chromosomes of CHO K1 cell line and 94 clones on primary Chinese hamster chromosomes to investigate the chromosome rearrangements. The karyotypic comparison between CHO DG44 and primary Chinese hamster cells was diagrammatically summarized in Fig. 1. The 20 chromosomes in CHO DG44 cell line were aligned in order of decreasing size and assigned letters from A to T. The normal Chinese hamster chromosome number were estimated using BAC-FISH, end-sequencing and previous comparative study between mouse and Chinese hamster [11]. The chromosomes A, B, C, F, L, N, R and S were derived from normal Chinese hamster chromosomes without large rearrangements, and then these chromosomes were conserved between CHO DG44 and K1 cells (data not shown). This result suggested that these chromosomes were very stable and essential in CHO cells and supposedly conserved in other CHO cell lines.

Figure 1
figure 1

The physical map of CHO DG44 cells and karyotypic comparison to primary Chinese hamster cells on the basis of BAC-FISH results. Homologous region of CHO DG44 to Chinese hamster were colored according to the estimated Chinese hamster chromosomes.

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FISH analysis of gene-amplified chromosomal region of transfected cells [8]

The chromosomal site of integration of a transgene affects its transcription rate. This phenomenon is called a positive effect and is often observed in transgenic organisms in which the transcription of an inserted transgene is affected by the proximity of the transgene to heterochromatin [12]. To understand the effect of structure of Dhfr amplicon on the chromosomal site of integration, we performed two-color BAC-FISH analysis for transgene. In our previous study, we determined that the amplified gene is integrated in one specific chromosome (chromosome O). The constructed vectors B and C contain the palindrome structure obtained from the BAC clone Cg0031N14 located on chromosome O. The summarized results of integrated chromosomal sites under various MTX concentrations are shown in Table 1. Interestingly, the transfected cells whose transgene is located at the same chromosomal position of the BAC clone Cg0160E04 were abundantly observed in the vector B and C integrations. It is likely that the chromosomal position of the BAC clone Cg0160E04 on chromosome O is a hot spot for Dhfr amplification in CHO cells.

Table 1 Ratios of amplified genes located at same position of BAC clone Cg0160E04 on chromosome O.
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In summary, we constructed a BAC-based physical map for CHO DG44 cells and analyzed genome-wide rearrangements of chromosome among CHO cells. This BAC-based physical map will greatly facilitate the studies of CHO cell genome. The BAC clones comprising this physical map could also provide a genome-wide resource for analysis of chromosome rearrangements, chromosome structure, comparative genome hybridization, gene targeting, and functional genomics.