Figure 4 | Scientific Reports

Figure 4

From: An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

Figure 4

Analysis of EGFP expression during long-term culture of CHO K1R EGFP MAR-HAC clones. (A,B) Western blotting following in cell lysates from PDL 3 and 50 of long-term cultures. Expression level of EGFP was normalized to tubulin. Expression of protein at PDL 3 was arbitrarily assigned as 1. Panel (A) shows CHO K1R EGFP MAR-HAC cl.1. Panel (B) shows CHO K1R EGFP MAR-HAC cl.2. Cropped blots were used in this figure. Original full-length blots are presented in Supplementary Figs S4, S5. Data shown are representative of at least three independent experiments. (C,D) Expression of EGFP was tracked in living cells with fluorescence microscopy. A representative photo of cells is shown. Scale bars: 100 μm. Panel (C) shows CHO K1R EGFP MAR-HAC cl.1. Panel (D) shows CHO K1R EGFP MAR-HAC cl.2. (E) Mitotic stability of the EGFP MAR-HAC vector in CHO K1 cells. Two-color FISH analysis of the CHO K1R EGFP MAR-HAC clones at PDL 50 was performed with digoxigenin-labeled hCot-1 DNA (red) and biotin-labeled EGFP (green). EGFP probe-specific green signals are indicated by the small arrowheads. The left panel shows CHO K1R EGFP MAR-HAC cl.1. The right panel shows CHO K1R EGFP MAR-HAC cl.2.

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