title: Enhanced green fluorescent protein as an efficient reporter gene for retroviral transduction of human multipotent lymphoid precursors.
abstract: Owing to its autofluorescence properties, green fluorescent protein (GFP) has aroused increasing interest as a marker system for many research applications. In this study we investigated the suitability of the "enhanced" GFP (EGFP), a mutant version of GFP optimized for flow cytometry and microscopy detection, as a reporter gene for retroviral transduction protocols. EGFP was shown to display a bright and stably maintained emission pattern in transfected GP+envAm12 packaging cells. Stable fluorescent emission was observed as well after transduction in NIH 3T3 fibroblasts and in the human Jurkat T cell line, in which EGFP was shown to confer no deleterious effect or growth disadvantage on the expressing cells. Moreover, EGFP expression could be detected after short-term retroviral exposure, thus allowing a rapid and quantitative retroviral titering assay, alternative to the standard colony-formation procedure. Most importantly, we showed the feasibility of EGFP as a marker gene in retroviral-mediated transduction of primary lymphoid precursors. In particular, transduction of CD34+CD1- human thymocytes by short-term cocultivation yielded up to 30% of EGFP-expressing cells, while maintaining CD34 expression levels. Finally, when cultured under multicytokine-supported conditions, such transduced intrathymic progenitors were shown to efficiently generate lymphoid-related dendritic cells, which displayed a distinct EGFP expression. Therefore, because of its rapid and easy detectability and its nontoxic characteristics, EGFP proves itself to be a valuable reporter gene by allowing the transduction of multipotential progenitors and by being compatible with the developmental programs of lymphoid lineage generation.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, Spain.
authors.forename: A R
authors.initials: AR
authors.lastname: Ramiro
authors.forename: V G
authors.initials: VG
authors.lastname: De Yébenes
authors.forename: C
authors.initials: C
authors.lastname: Trigueros
authors.forename: Y R
authors.initials: YR
authors.lastname: Carrasco
authors.forename: M L
authors.initials: ML
authors.lastname: Toribio
mesh_terms: D016475:3T3 Cells; D000818:Animals; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics*; D008214:Lymphocytes / Q000166:cytology*; D051379:Mice; D013997:Time Factors; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins