A selective method for transfection of retinal ganglion cells by retrograde transfer of antisense oligonucleotides against kynurenine aminotransferase II.
OBJECTIVE
Intravitreal administration of specific antisense oligonucleotides (ODNs) effectively downregulates gene expression in the retina but does not modulate it exclusively in retinal ganglion cells (RGCs). Expression of kynurenine aminotransferase II (KAT II) in RGCs has been well described in the literature. We describe a new method for downregulating cellular KAT II expression via transfection of RGC by retrograde transfer of ODN.


METHODS
Fluorescently labeled, specific ODNs against KAT II were injected into rats either intravitreally or into the superior colliculi. Fluorescence microscopy of retinal flat-mounts and radial sections was used to compare the location, duration, and degree of transfection for both methods of delivery. The effects of both methods on KAT II expression in RGCs were studied immunohistochemically with unlabeled ODN. Retinal kynurenic acid (KYNA) contents were measured using high pressure liquid chromatography (HPLC).


RESULTS
After intravitreal injection, fluorescently labeled ODN reached all retinal layers, whereas injections into the superior colliculus resulted in transfection of the RGC layer alone. Immunohistochemistry showed that both methods of ODN application had a similar effect on downregulation of KAT II expression in RGC. Retinal KYNA content decreased significantly 4 days after both types of ODN administration.


CONCLUSIONS
This study demonstrated that retrograde transfer of specific ODN into RGC is feasible and induces downregulation of KAT II cellular expression. This may become a useful tool for modulating gene expression in the retinal ganglion cell layer in vivo without direct transfer of ODN to other retinal cell layers.
Thaler
Sebastian
S
Department of Pathophysiology of Vision and Neuro-Ophthalmology, University Eye Hospital, Tübingen, Germany. sebastian.thaler@uni-tuebingen.de
Rejdak
Robert
R
Dietrich
Karen
K
Ladewig
Thomas
T
Okuno
Etsuo
E
Kocki
Tomasz
T
Turski
Waldemar Andrzej
WA
Junemann
Anselm
A
Zrenner
Eberhart
E
Schuettauf
Frank
F
D000818:Animals; D001693:Biological Transport, Active; D002232:Carbocyanines; D002851:Chromatography, High Pressure Liquid; D015536:Down-Regulation; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D007267:Injections; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D011914:Rats, Inbred BN; D012165:Retinal Ganglion Cells / Q000378:metabolism*; D013194:Staining and Labeling; D013266:Stilbamidines; D013477:Superior Colliculi; D013997:Time Factors; D000637:Transaminases / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D014822:Vitreous Body
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D002232:Carbocyanines; D005456:Fluorescent Dyes; D016376:Oligonucleotides, Antisense; D013266:Stilbamidines; C089187:cyanine dye 3; D000637:Transaminases; C021481:kynurenine-oxoglutarate transaminase