Development of Mammalian Cell Lines with lac Operator-Tagged Chromosomes

INTRODUCTIONThe discovery and use of fluorescent proteins to label chromosomal proteins has yielded basic structural information as well as insights into dynamics that were previously inaccessible. This protocol describes a method for tagging specific chromosome sites using the lac operator/repressor system, wherein direct repeats of bacterial operator sequences are coupled with green fluorescent protein (GFP)-tagged proteins that recognize these sequences. Direct lac operator repeats are generated by directional cloning. Although the use of direct repeats, as opposed to inverted repeats, reduces recombination within the bacterial host at high copy number, even the direct repeats are unstable, requiring the use of special bacterial hosts and low-copy-number plasmids for cloning. The introduction of the lac operator repeats into eukaryotic cells uses traditional transformation methods. Techniques for the isolation of stable cell clones with varying transgene copy numbers are described in the protocol, as are several methods for visual screening of large numbers of stable cell clones to isolate rare clones containing labeled chromosomal regions with desired features.