Fluorescence In Situ Hybridization (FISH)

FISH analysis was performed as following. Hamster CHO cells carrying the alphoid^tetO-HAC bearing the platform cassette A037 were cultured in F12 medium with 10 μg/mL of colcemid (Invitrogen) overnight at 37 °C. Metaphase cells were trypsinized and centrifugated for 4 min at 172g, treated in 10 mL of 50 mM KCl hypotonic solution for 20 min at 37 °C and washed three times in methanol:acetic acid (3:1) solution with a 4 min centrifugation at 172g between each wash. Cells were diluted to the appropriate density with fixative solution, spread onto precleaned slides (Thermo Fisher Scientific, Waltham, MA, USA) above steam (boiling water), and allowed to age 2 days at room temperature. For BAC probing, CHO metaphase slides were washed in 70% formamide in 2× SSC for 2 min at 72 °C. Samples were dehydrated through a 70, 90, and 100% ethanol series for 4 min each and left to air-dry. The probe used for FISH was BAC32–2-mer(tetO) DNA containing 40 kb of alphoid-tetO array cloned into a BAC vector as described previously.¹¹ BAC DNA was labeled using a nick-translation kit with Orange 552 dUTP (5-TAMRA-dUTP) (Abbott Molecular). The probe was denatured in hybridization solution at 78 °C for 10 min and left at 37 °C for 30 min. The hybridization mix probe was applied to the sample and incubated at 37 °C overnight. Slides were washed with 0.4× SSC, 0.3% Tween 20 for 2 min at 72 °C, briefly rinsed with 2× SSC, 0.1% Tween 20 (10 s) and air-dried in darkness. The samples were counterstained with VECTASHIELD mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA). Slides were analyzed by fluorescence microscopy. Images were captured using a DeltaVision imaging system in the CRC, LRBGE Fluorescence Imaging Facility (NIH) and analyzed using ImageJ software (NIH).