Adenovirus vector-directed expression of the neurotrophin-3 receptor (TrkC) in mouse astrocytes.
In the present article we report the generation of a neurovirological reagent, an adenovirus vector that efficiently delivers the gene for the Neurotrophin-3 (NT-3) receptor, TrkC. Using this AdTrkC vector, we examined the induction of the expression of the above neurotrophin receptor in pure cultures of mouse astrocytes, a glial cell type that does not constitutively express this gene. Infection of astrocytes at an optimal dose of 100-200 plaque forming units (p.f.u.) per cell, induced expression of specific mRNA, as demonstrated by RT-PCR and Northern blot. This mRNA was translated to produce a mean of 20,157 biologically active receptor molecules per astrocyte with a Kd of 4.1 x 10(-11) M, as demonstrated by 125I-NT-3 binding. After 2D electrophoresis, the mature glycoprotein and some precursors were recognised by antibodies raised against the carboxy-terminal peptide of Trk. Binding of the ligand induced autophosphorylation ofTrkC and 3H-thymidine incorporation in transduced cells. These results demonstrate that our AdTrkC vector efficiently mediates the expression of high-levels of biologically active NT-3 receptors.
Rubio
N
N
Department of Neuroimmunology, Instituto Cajal, Madrid, Spain.
Abad-Rodriguez
J
J
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007457:Iodine Radioisotopes; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D020933:Neurotrophin 3 / Q000378:metabolism / Q000493:pharmacokinetics; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D020812:Receptor, trkC / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D007457:Iodine Radioisotopes; D020933:Neurotrophin 3; D012333:RNA, Messenger; D004247:DNA; D020812:Receptor, trkC