title: Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo.
abstract: We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.
authors.affiliation: Department of Hematology/Oncology, St Jude Children's Research Hospital, Memphis, TN 38105-2794, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Persons
authors.forename: J A
authors.initials: JA
authors.lastname: Allay
authors.forename: E R
authors.initials: ER
authors.lastname: Allay
authors.forename: R J
authors.initials: RJ
authors.lastname: Smeyne
authors.forename: R A
authors.initials: RA
authors.lastname: Ashmun
authors.forename: B P
authors.initials: BP
authors.lastname: Sorrentino
authors.forename: A W
authors.initials: AW
authors.lastname: Nienhuis
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006410:Hematopoiesis / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D012190:Retroviridae
chemical_list: D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins