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Cell culture and establishment of NIH 3T3 cell clones with integrated BAC or plasmid DNA

PC Pankaj Chaturvedi
BZ Binhui Zhao
DZ David L. Zimmerman
AB Andrew S. Belmont
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NIH 3T3 cells (CRL-1658, ATCC) were routinely grown in Dulbecco’s modified Eagle medium (Life technologies) supplemented with 10% Bovine Growth Serum (BGS) (HyClone) and 1X Antibiotic-Antimycotic (Life Technologies). Purified GAPDH BAC DNA was linearized using the PI-SceI homing endonuclease and transfected into NIH 3T3 cells using Lipofectamine 2000 (Life technologies) according to the manufacturer’s directions. The reporter plasmid pUGG was linearized with NdeI and gel purified prior to transfection. Mixed clonal populations of stable transformants were obtained after 2 weeks of 75 μg/ml zeocin (Life Technologies) selection; individual cell clones were obtained by serial dilution or retrieval of colonies using filter discs.62

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