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. 2003 Jul 7;162(1):23–35. doi: 10.1083/jcb.200303098

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Copyright © 2003, The Rockefeller University Press

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Figure 1. — Experimental approach. (A) A pSVII-dhfr vector derivative was used to create control and dSAR constructs with 32 copies of a lac operator 8-mer repeat. (B) DHFR (−) CHO cells (DG44) were stably transfected with either control or dSAR supercoiled DHFR expression vectors. fluorescein-labeled MTX–stained cells with larger inserts were selected with FACS^® and cloned. (C) Mitotic chromosomes were isolated and used for extraction and staining by different methods; lac repressor immunostaining or in vivo expression of an EGFP–lac repressor–NLS fusion protein and FISH. (D) Cell flow cytometry (abscissa, logarithm of relative intensity; ordinate, cell number) after staining with fluorescein-labeled MTX. Average DHFR expression is ∼20× higher in cells transformed with the dSAR versus control vector.