Efficient gene transfer into primary and immortalized human fetal glial cells using adeno-associated virus vectors: establishment of a glial cell line with a functional CD4 receptor.
Adeno associated virus (AAV) is a non-pathogenic dependent parvovirus with a broad host range, capable of high levels of transduction and stable integration into the host cell genome. We have investigated the potential for using AAV as a vector for gene transfer into glial cells of the human fetal nervous system. Recombinant AAV vectors expression either the reporter gene beta-galactosidase or a human CD4 receptor were able to transduce both primary glial cells of the human fetal nervous system and an SV40 immortalized human fetal glial cell line (SVG). No difference in transduction efficiency was observed between the primary cells and the cell line which in both cases was as high as 95%. Stable transfectants of the glial cell line expressing the CD4 receptor were selected. An SVG/CD4 expressing line was then established. The presence of the CD4 receptor was confirmed by immunohistochemistry, Westerm immuno-blotting and flow cytometric analysis. The CD4 receptor was shown to be functional by infection of the SVG/CD4 cell line with the human immunodeficiency virus (HIV). Upon infection, the SVG/CD4 cells produced 20-fold higher levels of the HIV intracellular core antigen P24 than the CD4 negative parental cells and in addition formed syncytia. The use of AAV vectors should prove useful in biological investigations of human glial cells and offers promise as a means of ex vivo and in vivo gene delivery.
Keir
S D
SD
Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
Miller
J
J
Yu
G
G
Hamilton
R
R
Samulski
R J
RJ
Xiao
X
X
Tornatore
C
C
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001921:Brain / Q000196:embryology; D015704:CD4 Antigens / Q000235:genetics*; D002460:Cell Line; D002469:Cell Separation; D000229:Dependovirus; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D009457:Neuroglia / Q000166:cytology*; D014162:Transfection / Q000379:methods
D000900:Anti-Bacterial Agents; D015704:CD4 Antigens; D005839:Gentamicins; C010680:antibiotic G 418