Figure 2

EGFP amplification on the HAC vector via the IR/MAR HAC system. (A) Schematic diagram of insertion of a plasmid encoding EGFP into the HAC vector. A circular targeting construct encoding EGFP, the non-functional neo gene, and the loxP site targeting the HAC vector are shown. Cre recombinase-catalyzed integration regenerates a functional neo gene. The resulting inserted allele is shown at the bottom. (B) The site-specific insertion event was detected in G418-resistant clones with genomic PCR analysis. A primer was designed to span the HAC insertion junction-specific site (indicated by a thin arrowhead in A). EGFP HAC and EGFP MAR-HAC clones indicate G418-resistant CHO DG44 clones. HAC: parental CHO DG44 cells including an empty HAC were used as a negative control. Cropped gels were used in this figure. Original full-length gels are presented in Supplementary Fig. S1. (C) Two-color FISH analysis of the HAC vector in G418-resistant CHO DG44 clones was performed with digoxigenin-labeled hCot-1 DNA (red) and biotin-labeled EGFP (green). The arrow indicates the HAC, and the inset shows enlarged images of the HAC (EGFP probe-specific green signals are indicated by the arrowheads). The left panel shows EGFP HAC control cells. The middle and right panels show EGFP MAR-HAC clones. (D) Relative copy number analysis of EGFP on the HAC vector with qPCR. Data were normalized to NV1. The copy number of EGFP in the EGFP HAC clone was arbitrarily at set as 1. Bars are the means ± SD of three independent experiments. (E) Expression levels of EGFP were analyzed with western blotting. An EGFP HAC clone was used as a control. Expression levels of EGFP were normalized to levels of tubulin. Expression level of protein in the EGFP HAC clone was arbitrarily assigned as 1. Cropped blots were used in this figure. Original full-length blots are presented in Supplementary Fig. S2. Data shown are representative of at least three independent experiments. (F) Expression of EGFP was tracked in living cells with fluorescence microscopy. A representative photo of cells is shown. Scale bars: 100 μm.