1. Cell lines: mouse LA9 cells containing a minichromosome, here designated LA9hprt-hgy+cells, and chicken DT40 cells, here designated DT40hprt+hyg- cells. See Note 1.
2. Medium and supplements: DMEM (Life Technologies), P-mercaptoethanol (Sigma), fetal calf serum (FCS; Sigma), chicken serum (Gibco-BRL), hygromycin B (Roche), demecolcine (colcemid; Sigma), and hypoxanthine-aminopterin-thymine (HAT; Sigma).
3. Chemicals and biochemicals: phosphate-buffered saline: PBS, pH 7.3, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4; 0.2% trypsin in Versene; cytochalasin B (Sigma); PEG 1500 (Roche); Percoll (Pharmacia); human Y alphoid satellite DNA either labelled with biotin or 32P; primer pairs:
P1 (5' -AACTTCATCAGTGTTACATCAAGG-3'
5' -TGTGGCATTTTGTTATGTGG-3') P2 (5' -AGGAGATGTCAGGACTATCAGC-3' 5' -TCCATCCAGCTGGTCATATT-3'); streptavidin Alexa Fluor 488 (Molecular Probes); and biotinylated antistreptavidin (Vector).
4. Plastics and equipment: tissue culture flasks, dishes, multiple-well plates, test tubes, polycarbonate tubes (40 mL; Fisher), Beckman J-20 centrifuge and JA-20 rotor (Beckman), pulsed-field gel electrophoresis (PFGE) system Rotaphor (Biometra), and Peltier Thermal Cycler (MJ Research).
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