FIG. 1.
Experimental scheme of complementation assay. We previously created CENP-H conditional knockout cell line #5-5 in which CENP-H expression is inactivated when tetracycline (tet) is added to culture medium. Various deletion derivatives of the CENP-H cDNA are cloned downstream of the CMV promoter (CMVpro) in the pEGFP vector. These constructs integrate at random sites in the genome of #5-5 CENP-H conditional knockout cells. Cells express both full-length CENP-H cDNA and mutant CENP-H. When tetracycline is added to culture medium, expression of full-length CENP-H is turned off, but mutant CENP-H is still expressed. If cells survive after addition of TET, the mutant CENP-H complements CENP-H function. We also investigated subcellular localization of mutant CENP-Hs. puror, puromycin resistance gene.