title: Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states.
abstract: The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis. © 2016 International Society for Advancement of Cytometry.
authors.affiliation: BIO5 Institute, University of Arizona, Tucson, Arizona.
authors.forename: Partha
authors.initials: P
authors.lastname: Samadder
authors.affiliation: BIO5 Institute, University of Arizona, Tucson, Arizona.
authors.forename: Ning
authors.initials: N
authors.lastname: Weng
authors.affiliation: BIO5 Institute, University of Arizona, Tucson, Arizona.|Department of Cellular and Molecular Medicine, University of Arizona, Tucson, Arizona.|University of Arizona Cancer Center, Tucson, Arizona.
authors.forename: Thomas
authors.initials: T
authors.lastname: Doetschman
authors.affiliation: Department of Cellular and Molecular Medicine, University of Arizona, Tucson, Arizona.|University of Arizona Cancer Center, Tucson, Arizona.|Department of Surgery, University of Arizona, Tucson, Arizona.
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Heimark
authors.affiliation: BIO5 Institute, University of Arizona, Tucson, Arizona.|University of Arizona Cancer Center, Tucson, Arizona.|School of Plant Sciences, University of Arizona, Tucson, Arizona.
authors.forename: David W
authors.initials: DW
authors.lastname: Galbraith
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D060846:Cell Engineering / Q000379:methods*; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D010179:Pancreas / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D000199:Actins; D006657:Histones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases
keywords: development; flow cytometry; gene expression; oncogenesis; sorting