title: Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles.
abstract: The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.
authors.affiliation: Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München; lahm@dhm.mhn.de.
authors.forename: Harald
authors.initials: H
authors.lastname: Lahm
authors.affiliation: Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München.
authors.forename: Stefanie A
authors.initials: SA
authors.lastname: Doppler
authors.affiliation: Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München.
authors.forename: Martina
authors.initials: M
authors.lastname: Dreßen
authors.affiliation: Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München.
authors.forename: Klaudia
authors.initials: K
authors.lastname: Adamczyk
authors.affiliation: Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München.
authors.forename: Marcus-André
authors.initials: MA
authors.lastname: Deutsch
authors.affiliation: Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München.
authors.forename: Hanna
authors.initials: H
authors.lastname: Ulrich
authors.affiliation: Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München; Clinical Cooperation Groups: "Antigen-specific Immunotherapy" and "Immune Monitoring", Helmholtz Center Munich (Neuhererg), Technische Universität München.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schiemann
authors.affiliation: Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München; DZHK (German Center for Cardiovascular Research) - Partner site Munich Heart Alliance.
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Lange
authors.affiliation: Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München; DZHK (German Center for Cardiovascular Research) - Partner site Munich Heart Alliance.
authors.forename: Markus
authors.initials: M
authors.lastname: Krane