Establishing isogenic inducible cell lines using founder reporter lines and recombinase-mediated cassette exchange.
Manipulating gene expression in mammalian cell lines is one of the most widely used methods for studying gene function. Tetracycline- and doxycycline-inducible systems are sensitive, reproducible, relatively inexpensive, and proven to work well in both cell lines and mouse models. However, obtaining homogeneous transgene expression or uniform knockdown by short hairpin RNA requires time-consuming and labor-intensive single-cell cloning to derive stable cell lines. For this reason, Tet-inducible cell systems have yet to be widely adopted. Here we describe the XT-cell method, a novel system for establishing isogenic inducible cell lines using founder reporter lines and recombinase-mediated cassette exchange. We demonstrate that, using this XT-cell method, isogenic stable Tet-inducible cell lines can be efficiently created with much less effort and time as compared with conventional methods. The XT-plasmids and the XT-founder cell lines will be a valuable resource to researchers interested in versatile modulation of gene expression in cell culture systems, and this method has the potential to expedite many aspects of biomedical research.
Departments of Pathology, Gynecology and Obstetrics, Johns Hopkins University School of Medicine, Baltimore, MD.
Bin
B
Guan
Tae
T
Magomi
Tian-Li
TL
Wang
Ie-Ming
IeM
Shih
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002460:Cell Line / Q000378:metabolism*; D004318:Doxycycline / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D045522:Recombinases / Q000235:genetics*; D013752:Tetracycline / Q000378:metabolism*; D019076:Transgenes
D000900:Anti-Bacterial Agents; D034741:RNA, Small Interfering; D045522:Recombinases; D013752:Tetracycline; D004318:Doxycycline