title: Murine Stem Cell-Based Retrovirus Production for Marking Primary Mouse Mammary Cells for Metastasis Studies.
abstract: Since the introduction of retroviral vector technology, permanent genetic marking of cells has considerably contributed to the understanding of different physiological and disease processes in vivo. Recent marking strategies aim to elucidate the contribution of cells on the clonal level, and the advent of fluorescent proteins has opened new avenues for the in vivo analysis of gene-marked cells. Gene-modified cells are easily identifiable (e.g., via the introduced fluorescent protein) within whole organ structures, allowing one to measure the contribution of transduced cells to malignant outgrowth. In our laboratory, we use the tetracycline-inducible system to study oncogene cooperation in metastatic progression. We use bicistronic retroviruses expressing the tetracycline transactivator (tTA) and the candidate gene (MIT-gene) or the tTA alone (MIT-Rx) to infect primary mammary cells from mice harboring tetracycline-inducible transgenes. This allows for constitutive expression of the candidate gene and tTA-dependent expression of the inducible oncogene. We also use MIG-based vectors, which allow for constitutive expression of the candidate gene and a green fluorescent protein. Here we describe how to produce retroviral particles carrying both MIT- and MIG-based vectors. Because of the fragility of the retroviral envelope, we do not attempt to concentrate the virus, and we directly use packaging cell media to infect primary epithelial cells (either normal or tumor). Infected cells can be transplanted into recipient mice to investigate metastatic colonization.
authors.affiliation: Department of Medicine, University of Louisville, Louisville, Kentucky 40202; Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky 40202;
authors.forename: Levi J
authors.initials: LJ
authors.lastname: Beverly
authors.affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Quebec H2W 1R7, Canada; Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Quebec H3T 1J4, Canada; Department of Medicine, Experimental Medicine Division, McGill University, Montréal, Quebec H3A 1A3, Canada.
authors.forename: Katrina
authors.initials: K
authors.lastname: Podsypanina
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D051379:Mice; D009362:Neoplasm Metastasis / Q000473:pathology / Q000503:physiopathology; D012190:Retroviridae / Q000235:genetics / Q000254:growth & development*; D013234:Stem Cells / Q000502:physiology*; D014161:Transduction, Genetic; D014776:Virus Cultivation