High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells.
Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.
Limón
A
A
Department of Cryobiology, Institut de Recerca Oncològica, Barcelona, Spain.
Briones
J
J
Puig
T
T
Carmona
M
M
Fornas
O
O
Cancelas
J A
JA
Nadal
M
M
García
J
J
Rueda
F
F
Barquinero
J
J
D016475:3T3 Cells; D000818:Animals; D018952:Antigens, CD34; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D012190:Retroviridae
D018952:Antigens, CD34; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins