Identification and isolation of human epithelial cell colonies that express specific gene products.
A new and simple method for the identification and isolation of human cell clones expressing specific gene products is presented. This technique is analogous to the colony blotting techniques described for prokaryotes. In this technique, human epithelial cells are grown in tissue-culture dishes, and cells from the colonies on the dish are transferred in situ to a high-density cationized quaternary amine-charged nylon membrane. The membrane can then be processed multiple times, using antibody and/or nucleic acid probes, for the identification of those cells producing the desired protein, mRNA or DNA sequence. Once identified, the desired cell colony is isolated for expansion, direct DNA sequencing and/or cell function. We demonstrate the potential of the technique by identifying and isolating colonies of replicating normal human liver hepatocytes producing albumin and keratin.
Gibson-D'Ambrosio
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Ohio State University Columbus, USA.
Brady
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D'Ambrosio
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D000418:Albumins / Q000032:analysis / Q000235:genetics*; D002460:Cell Line; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007633:Keratins / Q000032:analysis / Q000235:genetics*; D008099:Liver / Q000166:cytology* / Q000378:metabolism*; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D009757:Nylons; D012333:RNA, Messenger / Q000032:analysis
D000418:Albumins; D008567:Membranes, Artificial; D009757:Nylons; D012333:RNA, Messenger; D007633:Keratins