Enhanced green fluorescent protein as an efficient reporter gene for retroviral transduction of human multipotent lymphoid precursors.
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.
Ramiro
A R
AR
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Cantoblanco, Spain.
De Yébenes
V G
VG
Trigueros
C
C
Carrasco
Y R
YR
Toribio
M L
ML
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
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins