Figure 1.
The schematic structures are illustrated in this Figure. The black arrow represents gene and its orientation. An IR from DHFR non-coding region and an AR1 MAR from Ig κ intron are illustrated as dashed bold line and an oval respectively in D, G and H.
Figure 2.
Generation of several kinds of amplified structures by the IR/MAR plasmid depending on the cell type.
The IR/MAR plasmid was transfected into cells, a stable transformant was selected by drug selection, and a metaphase chromosome spread was prepared. Green fluorescence indicates the plasmid sequence after FISH and red fluorescence indicates the chromosome after propidium iodide (PI) staining. Representative images of several types of gene amplification are shown. In COLO 320DM cells, the plasmid was usually amplified as extrachromosomal DMs (A) or an chromosomal homogeneous array of HSR (B). In CHO DG44 cells (C to H), the IR/MAR plasmid was amplified as a chromosomal structure of varying length, including a tiny dot (C), a line (D) and a HSR longer than the metaphase chromosome width (cw), which appeared as either a fine ladder (E to G) or a ladder (H) structure. For the definition of these structures, see the text. The frequencies of these structures were counted and shown in the graphs appearing in Figures 3 to 5 using the legend shown in panel I of this Figure.
Figure 3.
Efficient antibody production by the IR/MAR gene amplification method.
COLO 320DM cells (A), CHO DG44 cells (B) and CHO-K1 cells (C) were transfected with IR/MAR-bearing pBM-MycLH or the control IR/MAR-negative pV-MycLH plasmid. CHO DG44 cells were co-transfected with IR/MAR-bearing pΔBN.AR1 and pMycLH plasmids (D, E), or co-transfected with control pSFV-V and pMycLH (D, F) plasmids. After selection of the stable transformant for about one month, gene amplification (A to C, D) and antibody expression (A’ to D’) were examined by FISH and ELISA, respectively. Clones were obtained from the cells shown in D by the limiting dilution in 96 well plates. The wells bearing a single colony were microscopically determined, and the culture liquid was analyzed by ELISA. The horizontal axis of panel E or F corresponds to 43 or 53 clones, respectively.
Figure 4.
Effect of the plasmid construct and method of transfection.
(A) The promoter sequences noted in the Figure were inserted on the 5′-sides of the Ig genes (MycH and MycL) in the constructs shown in Fig. 1A and B, and each of these plasmids was co-transfected with the IR/MAR-bearing plasmid (pΔBN AR1). (B) pBM-MycLH forward (FW) or reverse (REV), for which the orientations of the Ig gene were different, was also transfected. (C) Transfection with the three combinations of plasmids noted in the Figure. The plasmid was transfected to CHO DG44 cells, and the polyclonal transfectants were selected by blasticidine for about one month. The amplified structure (B, C) and the protein expression level (A, B’ and C’) were examined as in Fig. 3.
Figure 5.
Clonal stability with respect to antibody expression and amplified genes.
Two high-producer clones with amplified genes at a chromosomal line HSR (A; CN19-10/1A3) or a ladder HSR (B; CN19-4/1F3) were subjected to prolonged culture. The antibody expression in the presence or absence of butyrate (A, B), the copy number of the Ig-light chain (MycL) and the IR sequence per cell (A’, B’), and the amplified structure were examined by ELISA, real-time PCR and FISH, respectively, at the days after the plasmid transfection that were noted in the graph.
Figure 6.
The integrity and the reactivity of the antibody produced by this method.
SDS-PAGE of proteins produced by clone CN19-4/1F3. lane1, antibody purified from the clone CN19-4/1F3 culture; lane 2, commercial 9E10 c-MYC antibody; lane 3, immunoblot of the 50 kDa c-MYC fusion protein with the purified antibody from the CN19-4/1F3 culture (lane 3) or the commercial 9E10 c-MYC antibody (lane 4).
Figure 7.
Production of antibody in suspension serum-free culture.
The high producer cell clones (A, CN19-10/1A3; B, CN19-4/1F3) were adapted to suspension serum-free culture as described in the Materials and Methods section. The cell density and the antibody concentration in the medium are shown in the Graphs. The arrows indicate the time points at which the entire medium was changed.