title: An inducible Tet-Off-H2B-GFP lentiviral reporter vector for detection and in vivo isolation of label-retaining cells.
abstract: Many regenerative cells are label-retaining cells (LRCs) due to their ability to keep a DNA label over a prolonged time. Until recently, isolation of vital LRCs was hampered due to the necessary use of fixation methods. To circumvent this, we generated a lentiviral-(HIV-1) based vector expressing a Tet-Off controlled histone 2B-GFP (Tet-Off-H2B-GFP) reporter gene for the detection and isolation of viable LRCs. In initial experiments, the vector was successfully used to infect 2- and 3-dimensional tissue culture models. Infected cultures from skin and pancreatic cells showed a very tight regulation of H2B-GFP, were sensitive to minimal amounts of doxycycline (Dox) and had a stable transgenic expression over the time of this study. Our lentiviral vector represents a reliable and easy to handle system for the successful infection, detection and isolation of LRCs from various tissues in vitro, in vivo and ex vivo.
authors.affiliation: Genetics of Skin Carcinogenesis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Berit
authors.initials: B
authors.lastname: Falkowska-Hansen
authors.forename: Jasmin
authors.initials: J
authors.lastname: Kollar
authors.forename: Barbara Maria
authors.initials: BM
authors.lastname: Grüner
authors.forename: Merle
authors.initials: M
authors.lastname: Schanz
authors.forename: Petra
authors.initials: P
authors.lastname: Boukamp
authors.forename: Jens
authors.initials: J
authors.lastname: Siveke
authors.forename: Axel
authors.initials: A
authors.lastname: Rethwilm
authors.forename: Marc
authors.initials: M
authors.lastname: Kirschner
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006657:Histones / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection
chemical_list: D006657:Histones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004318:Doxycycline