Expression of heterologous proteins in cultured rat hippocampal neurons using the Semliki Forest virus vector.
The Semliki Forest virus expression vector (Liljeström and Garoff: Bio/Technology 9:1356-1361, 1991) was tested in cultured rat hippocampal neurons using two Madin-Darby canine kidney (MDCK) cell membrane-associated proteins as reporters: rab8, a small GTPase involved in post-Golgi vesicle transport, and VIP21, an integral membrane protein of caveolae, trans-Golgi network, and post-Golgi vesicles. Expression of the c-myc epitope-tagged proteins was visualized by immunofluorescence microscopy. The proteins were first detected in neurons after 3-4 hr infection by the recombinant viruses. The infection efficiency on neurons was high: after 6 hr infection at a multiplicity of one, 50-60% of the cells expressed the reporter proteins. The neurons tolerated the infection well up to 8 hr. Their polarized organization was not disturbed, as judged from morphology and from distribution of the dendritic MAP2 and axonal synaptophysin marker proteins. The Semliki Forest virus vector thus seems suitable for short-term expression of proteins in cultured neurons.
Olkkonen
V M
VM
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Liljeström
P
P
Garoff
H
H
Simons
K
K
Dotti
C G
CG
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004285:Dogs; D005260:Female; D005455:Fluorescent Antibody Technique; D020558:GTP Phosphohydrolases / Q000096:biosynthesis / Q000235:genetics; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D005822:Genetic Vectors; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D009474:Neurons / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012672:Semliki forest virus / Q000235:genetics*; D013347:Subcellular Fractions / Q000378:metabolism; D020691:rab GTP-Binding Proteins
D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D004247:DNA; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; C082377:RAB8A protein, human; C513177:Rab8b protein, rat; D020691:rab GTP-Binding Proteins