Functional analysis of bacterial artificial chromosomes in mammalian cells: mouse Cdc6 is associated with the mitotic spindle apparatus.
Bacterial artificial chromosomes (BACs) provide a well-characterized resource for studying the functional organization of genes and other large chromosomal domains. To facilitate functional studies in cell cultures, we have developed a simple approach for generating stable cell lines with variable copy numbers of any BAC. Here we describe hamster cell lines with BAC transgenes that express mouse Cdc6 at levels that correlate with BAC copy number; show that mouse Cdc6 is regulated normally during the cell cycle, binds chromatin, and is degraded during apoptosis; and report a novel fraction of Cdc6 that associates with the spindle apparatus during mitosis. With RNA interference to assess genetic complementation by BAC alleles, this system will facilitate functional studies on large chromosomal domains at variable copy number in mammalian cell models.
Illenye
Sharon
S
Department of Pathology and Vermont Cancer Center, University of Vermont College of Medicine, Burlington 05405, USA.
Heintz
Nicholas H
NH
D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D002843:Chromatin / Q000378:metabolism; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D015151:Immunoblotting; D051379:Mice; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D019076:Transgenes / Q000235:genetics
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D034741:RNA, Small Interfering; D029701:Saccharomyces cerevisiae Proteins