Efficient gene transfer in mouse neural precursors with a bicistronic retroviral vector.
Gene transfer into neural precursors is a powerful approach to study the function of specific gene products during nervous system development. Here we describe a retrovirus-based methodology to transduce foreign genes into mouse neural precursors. We used a high-titer bicistronic retroviral vector that encodes a marker gene, placental alkaline phosphatase (plap), and a selection gene, neomycin phosphotransferase II (neoR), under the translational control of two retroviral internal ribosome entry segments. Transduction efficiency even without selection was up to 95% for multipotential neurospheres derived from embryonic striata and grown with basic fibroblast growth factor 2. Expression of plap and neoR was sustained with time in culture and upon differentiation into neurons, astrocytes, and oligodendrocytes, as shown by double immunofluorescence labeling with cell type-specific markers, Western blotting, and neomycin resistance. However, levels of plap were decreased in differentiated oligodendrocytes. Transduction with the same vector of neonatal oligodendrocyte precursors grown in oligospheres consistently resulted in a lower proportion of plap-immunoreactive cells and enhanced cell death in the absence of neomycin. However, plap expression was maintained in some differentiated oligodendrocytes expressing galactocerebroside or myelin basic protein. In that neurospheres can be easily expanded in vitro and factors enabling their differentiation into the three main central nervous system cell types are being elucidated, this methodology could be used in the future to produce large number of transduced, differentiated neural cells.
Franceschini
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IA
Unité de Neurovirologie et Regénération du Système Nerveux, Pasteur Institute, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Feigenbaum-Lacombe
V
V
Casanova
P
P
Lopez-Lastra
M
M
Darlix
J L
JL
Dalcq
M D
MD
D000469:Alkaline Phosphatase; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D003342:Corpus Striatum / Q000166:cytology* / Q000196:embryology; D003673:Defective Viruses / Q000235:genetics*; D004351:Drug Resistance; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D058851:GPI-Linked Proteins; D015870:Gene Expression; D005796:Genes; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D005839:Gentamicins / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D019868:Kanamycin Kinase / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009836:Oligodendroglia / Q000166:cytology / Q000378:metabolism; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D001356:Reticuloendotheliosis virus / Q000235:genetics*; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes
D058851:GPI-Linked Proteins; D005839:Gentamicins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; C010680:antibiotic G 418; D019868:Kanamycin Kinase; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental