Figure 1.
FcR plasmids and expression of the FcR gene.
CHO DXB-11 cells were transfected with the IR/MAR-positive pΔBM AR1Dhfr-FcR plasmid or the IR/MAR-negative pECE-FcR Dhfr plasmid [23_ENREF_23] by electroporation, and then cultured for 10 days in α-MEM(−). Clones were obtained by limiting dilution; the highest producer of FcR was determined by ELISA and then cultured in the presence of 25 nM Mtx for 15 days. Surviving cells were subjected to another round of limiting dilution and the highest producers were then cultured in the presence of 125 nM Mtx for 20 days. The same selection process was used for clones obtained using the conventional Dhfr/Mtx method; however, cells were selected and cloned in medium containing 5, 50 and 500 nM Mtx. After each round of selection, the level of FcR protein (µg/ml) was determined by ELISA.
Figure 2.
In panels A to K, plasmid sets α to δ are labeled in red; (+) and (−) indicate the presence and absence of IR/MAR, respectively. Double-headed red arrows indicate the co-transfected plasmid set α and γ. Double-headed black arrows indicate the two sets of co-transfected plasmids used to quantify GFP expression by flow cytometry (Figure 3). In each plasmid, blue arrows represent the target genes (d2EGFP, MycL, and MycH) and green arrows represent the drug-resistance genes (Dhfr, BS, and Neo); the IR and MAR are represented by a red dashed bold line and red oval, respectively. The promoters (P-CMV, P-SRα or P-SV40), polyA-addition sequences (TK-pA or SV40-pA), and sequences required for cloning in E. coli (Ori and Amp), are shown in gray. In panel L, the features of the plasmids are summarized. The font colors are the same as those used in panels A to K.
Figure 3.
Quantitation of d2GFP gene expression by flow cytometry.
CHO DG44 cells were transfected with the pCMV-d2EGFP plasmid and IR/MAR-negative pSFV-V-Dhfr (A, C) or IR/MAR-positive pΔBN AR1-Dhfr (B, D) plasmid. These vector sets are indicated by the double-headed black arrows in Figure 2. Cells were then selected with 10 µg/ml blasticidin in αMEM(+)(A and B), or with 10 µg/ml blasticidin and 5 nM Mtx in αMEM(−) (C and D). Stable transfectants produced without gene amplification (A), with IR/MAR-amplification (B), Dhfr/Mtx amplification (C), or IR/MAR-Dhfr fusion amplification (D), were obtained and d2GFP fluorescence was detected using flow cytometry. The number of culture days at the time of analysis is noted in each panel.
Figure 4.
Cytogenetic appearance of amplified sequences.
FISH analysis of metaphase spreads prepared from transfected CHO DG44 cells showing dots (A); lines (B); a fine ladder HSR of approximately two (C, I, and J), four (D), or six (E) chromosome widths (cw); and a ladder HSR (F). The frequencies of these structures were determined and are shown in Figures 5 to 7. Multiple HSRs in a single cell (G) or ring chromosomes consisting of ladder HSRs (H) were observed in transfectants grown in 500 nM Mtx. The densities of the plasmid signals in the HSR shown in panels I and J are clearly different; however, both were classified as two cw, because the signals are distributed along the length of two cws.
Figure 5.
Amplification and antibody expression using plasmid set
α. CHO DG44 cells were co-transfected with pMycLH (plasmid 2) and pΔBN AR1-Dhfr or pSFV-V-Dhfr (plasmid 1); cells were then selected by culture in the presence of 500 µg/ml G418, and the indicated concentrations of blasticidin (BS) and Mtx. The transfectant number (No.), Mtx concentration (nM), BS concentration (µg/ml), αMEM used (with (αMEM +) or without (αMEM−) nucleosides and deoxynucleosides), and amplification method, are indicated at the bottom of the figure. IR/MAR: IR/MAR method; Conv: conventional expression plasmid. Cells reached confluence at the indicated number of days after transfection (Days a Trf). Cytogenetic structures were analyzed by FISH (A) (cw: chromosome width). Antibody expression was quantified by real-time PCR (B), and ELISA (C). Cells prepared for ELISA were grown in the presence (+) or absence (−) of 10 mM sodium butyrate (But). Error bars represent mean +/− S.D.
Figure 6.
Amplification and antibody expression using plasmid sets ß and γ.
CHO DG44 cells were transfected with plasmids 1 and 2 (as indicated) of sets β (A to C) and γ (D to F), and then selected by culture in the presence of 10 µg/ml BS, the indicated concentrations of Mtx, and the absence of G418. The transfectant number (No.), Mtx concentration (nM), and the amplification method, are indicated at the bottom of the figure. Cells reached confluence at the indicated number of days after transfection (Days a Trf). Cytogenetic structures were analyzed by FISH (A) (cw: chromowome width). Antibody expression was quantified by real-time PCR (B), and ELISA (C). Cells prepared for ELISA were grown in the presence (+) or absence (−) of 10 mM sodium butyrate (But). Error bars represent mean +/− S.D.
Figure 7.
Amplification and antibody expression by using plasmid set δ.
Plasmids 1 (indicated) of plasmid set δ were co-transfected and selected by culture in the presence of 10 µg/ml blasticidin, the indicated concentrations of Mtx, and the absence of G418. The transfectant number (No.), Mtx concentration (nM), and the amplification method are indicated at the bottom of the figure. Transfectants CN61-3 and -1, and CN61-5 and -6, differ by whether the selection was started from 0 or 5 nM Mtx in the nucleotide-deficient medium. Cells reached confluence at the indicated number of days after transfection (Days a Trf). Cytogenetic structures were analyzed by FISH (A) (cw: chromowome width). Antibody expression was quantified by real-time PCR (B), and ELISA (C). Cells prepared for ELISA were grown in the presence (+) or absence (−) of 10 mM sodium butyrate (But). Error bars represent mean +/− S.D.
Figure 8.
Clones obtained from plasmid set δ.
Polyclonal transfectants (CN61-1, CN61-3, CN61-5, and CN61-6) that had been adapted to 0 nM (M0) or 5 nM (M5) Mtx in nucleotide-deficient medium (Figure 7), and polyclonal transfectant CN49-4 (Figure 5), were subjected to limiting dilution in 96-well plates. Wells containing a single colony were recorded and the culture medium from 100 (CN61-1, CN61-3, CN61-5, and CN61-6) or 50 (CN49-4) colonies for each transfectant was analyzed by ELISA (1st screening; data not shown). The ten (CN61-1, CN61-3, CN61-5, and CN61-6) or five (CN49-4) highest antibody-producing clones of each transfectant were selected; these cells were cultured in 96-well plates for 3 days in the presence or absence of 10 mM butyrate and the culture medium was analyzed by ELISA. The mean +/− S.D. are indicated in the upper right corner of each panel. Error bars represent mean +/− S.D.
Figure 9.
Stability of clones obtained using plasmid set δ.
The five highest antibody-producing plasmid set δ clones generated using the IR/MAR-fusion (A to E) and Dhfr/Mtx (F to J) methods were selected and cultured in the presence (red lines) or absence (blue lines) of selective pressure (10 µg/ml blasticidin and 5 nM Mtx in the absence of nucleotides). After the indicated number of days post-transfection, cells were cultured in 96-well plates for 3 days in the presence (solid lines) or absence (dashed lines) of 10 mM butyrate and the culture medium was analyzed by ELISA.