Direct gene transfer in the Gottingen minipig CNS using stereotaxic lentiviral microinjections.
We aim to induce direct viral mediated gene transfer in the substantia nigra (SN) of the Gottingen minipig using MRI guided stereotaxic injections of lentiviral vectors encoding enhanced green fluorescent protein (EGFP). Nine female Gottingen minipigs were injected unilaterally into the SN with 6 per 2.5 microliters lentivirus capable of transducing cells and mediating expression of recombinant EGFP. The animals were euthanized after four (n=3) or twenty weeks (n=6). Fresh brain tissue from three animals was used for PCR. The remaining six brains were cryo- or paraffin-sectioned for fluorescence, Nissl-, and immunohistochemical EGFP visualization. EGFP was seen in nigral neurons, axons, glial cells, endothelial cells, and in nigral fibers targeting the striatum. PCR-based detection confirmed the presence of the transgene in SN, whereas all other examined brain areas were negative, indicating that the immunohistochemically detected EGFP in the striatum derived from transfected nigral cells.
Norgaard Glud
Andreas
A
Institute of Anatomy, Faculty of Health Sciences, University of Aarhus, DK-8000 Aarhus C, Denmark. angl@ana.au.dk.
Hedegaard
Claus
C
Nielsen
Mette Slot
MS
Sørensen
Jens Christian
JC
Bendixen
Christian
C
Jensen
Poul Henning
PH
Larsen
Knud
K
Bjarkam
Carsten Reidies
CR
D000818:Animals; D001522:Behavior, Animal / Q000502:physiology; D001921:Brain / Q000821:virology; D004247:DNA / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics* / Q000254:growth & development; D008845:Microinjections; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013238:Stereotaxic Techniques; D013552:Swine; D013556:Swine, Miniature
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA