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2. Batches of FBS were tested for maximum single-cell cloning efficiency. FBS was added to medium immediately before use. Cultures were always passaged 1:4 at <80% confluence by brief treatment with 0.25% trypsin (without EDTA) and minimal pipetting. These conditions produce 30 to 50% single-cell cloning efficiency.
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HE and HO indicate heterozygous and homozygous derivatives, respectively. Nontargeted siblings are identified by clone number only. LF1 and HO7.2-1 cells tested negative for mycoplasma, human immunodeficiency virus, and HPV, and their karyotypes were normal.
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Passage number after gene targeting was arbitrarily set to 0 for each cell strain when the culture was first expanded into a 10-cm culture dish.
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We thank R. Halaban for establishing cell strains, H. Ozer and J. McCormick for advice on HDF culture, B. Vogelstein for the hyg p21 vector, and H.-F. Mark for karyotype analysis. Supported by grants from the Yale Skin Disease Research Center and the American Anti-vivisection Society (J.P.B.) and by NIH grant GM41690 (J.M.S).
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