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Non-viral siRNA delivery into the mouse retina in vivo.

Turchinovich A, Zoidl G, Dermietzel R - BMC Ophthalmol (2010)

Bottom Line: However, siRNA accumulation was predominantly confined to ganglion cells layer as analysed 24 h post-injection.Furthermore, siRNA containing particles were localized along GFAP cytoskeleton of retinal astroglial cells hinting on intracellular localization of the siRNA CONCLUSIONS: In this work we demonstrated that siRNA can be efficiently delivered into the vertebrate retina in vivo with low-toxicity using a non-viral carrier, specifically Transit-TKO transfection reagent.Our report could raise a closer look on Transit-TKO transfection reagent as a promising siRNA carrier in vivo and be of interest for the researchers and companies who work on development of ocular RNAi techniques.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum, Germany. a.turchinovich@dkfz.de

ABSTRACT

Background: Gene silencing in the retina using RNA interference could open broad possibilities for functional studies of genes in vivo and for therapeutic interventions in eye disorders. Therefore, there is a considerable demand for protocols to deliver siRNA into the vertebrate retina. In this work we explored a possibility to deliver synthetic 21 bp siRNA into the mouse retina after intravitreal application using a non-viral carrier.

Methods: Fluorescently labelled synthetic 21 bp siRNA duplex was combined with Transit-TKO transfection reagent and injected intravitreally into adult mice eyes. Eyes cryostat sections and whole mount retinas were prepared 24-48 h post-injection, stained with either Hoechst 33342 (cell nuclei) or immunostained with anti-GFAP antibody (astroglia cells marker). Distribution of fluorescent siRNA signal in the retina was investigated.

Results: Single intravitreal injection of as little as 5 ng of siRNA combined with Transit-TKO transfection reagent by a modified protocol provided robust and non-toxic delivery of the siRNA into the retina. However, siRNA accumulation was predominantly confined to ganglion cells layer as analysed 24 h post-injection. Furthermore, siRNA containing particles were localized along GFAP cytoskeleton of retinal astroglial cells hinting on intracellular localization of the siRNA CONCLUSIONS: In this work we demonstrated that siRNA can be efficiently delivered into the vertebrate retina in vivo with low-toxicity using a non-viral carrier, specifically Transit-TKO transfection reagent. However, the capacity of siRNA delivered by our protocol to induce gene silencing in the retina has to be further evaluated. Our report could raise a closer look on Transit-TKO transfection reagent as a promising siRNA carrier in vivo and be of interest for the researchers and companies who work on development of ocular RNAi techniques.

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Intravitreal injection of siRNA. A. Fluorescent micrograph of a cryostat section and B), C) whole mount retinas prepared 24 h after single intravitreal injection with 20 ng fluorescent siRNA (red) combined with Transit-TKO in low ionic environment. Note, the prominent localization of siRNA in the ganglion cells layer (GCL) of the retina. Arrows indicate the examples of cytoplasmic accumulation of siRNA near to cells nuclei. Low and highly transfected regions of the retina are marked by ovals (I, II). Cells nuclei were counterstained with Hoechst (blue). Scale bar = 20 μm.
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Figure 1: Intravitreal injection of siRNA. A. Fluorescent micrograph of a cryostat section and B), C) whole mount retinas prepared 24 h after single intravitreal injection with 20 ng fluorescent siRNA (red) combined with Transit-TKO in low ionic environment. Note, the prominent localization of siRNA in the ganglion cells layer (GCL) of the retina. Arrows indicate the examples of cytoplasmic accumulation of siRNA near to cells nuclei. Low and highly transfected regions of the retina are marked by ovals (I, II). Cells nuclei were counterstained with Hoechst (blue). Scale bar = 20 μm.

Mentions: After single intravitreal injection of as little as 20 ng of siRNA combined with Transit-TKO reagent robust transfection of the retina was observed. Fluorescent signals of siRNA were reproducibly detected in the ganglion cells layer (GCL) of the retina as aggregates of variable sizes 24 h post-injection (Fig. 1A). Significantly fewer aggregates were found in the inner plexiform and inner nuclear layers suggesting that siRNA uptake was limited to the innermost cell layer 24 h post injection. However, we did not analyze the dynamic of siRNA penetration into the retina in later time points. In sections of whole mount retina preparations a typical distribution of siRNA in the ganglion cells layer with fluorescent particles frequently located close to the cell nuclei was observed (see arrows; Fig. 1B). However, siRNA accumulation was not uniform along the retina, revealing regions of high and low transfection (Fig. 1C indicated by oval regions).


Non-viral siRNA delivery into the mouse retina in vivo.

Turchinovich A, Zoidl G, Dermietzel R - BMC Ophthalmol (2010)

Intravitreal injection of siRNA. A. Fluorescent micrograph of a cryostat section and B), C) whole mount retinas prepared 24 h after single intravitreal injection with 20 ng fluorescent siRNA (red) combined with Transit-TKO in low ionic environment. Note, the prominent localization of siRNA in the ganglion cells layer (GCL) of the retina. Arrows indicate the examples of cytoplasmic accumulation of siRNA near to cells nuclei. Low and highly transfected regions of the retina are marked by ovals (I, II). Cells nuclei were counterstained with Hoechst (blue). Scale bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959040&req=5

Figure 1: Intravitreal injection of siRNA. A. Fluorescent micrograph of a cryostat section and B), C) whole mount retinas prepared 24 h after single intravitreal injection with 20 ng fluorescent siRNA (red) combined with Transit-TKO in low ionic environment. Note, the prominent localization of siRNA in the ganglion cells layer (GCL) of the retina. Arrows indicate the examples of cytoplasmic accumulation of siRNA near to cells nuclei. Low and highly transfected regions of the retina are marked by ovals (I, II). Cells nuclei were counterstained with Hoechst (blue). Scale bar = 20 μm.
Mentions: After single intravitreal injection of as little as 20 ng of siRNA combined with Transit-TKO reagent robust transfection of the retina was observed. Fluorescent signals of siRNA were reproducibly detected in the ganglion cells layer (GCL) of the retina as aggregates of variable sizes 24 h post-injection (Fig. 1A). Significantly fewer aggregates were found in the inner plexiform and inner nuclear layers suggesting that siRNA uptake was limited to the innermost cell layer 24 h post injection. However, we did not analyze the dynamic of siRNA penetration into the retina in later time points. In sections of whole mount retina preparations a typical distribution of siRNA in the ganglion cells layer with fluorescent particles frequently located close to the cell nuclei was observed (see arrows; Fig. 1B). However, siRNA accumulation was not uniform along the retina, revealing regions of high and low transfection (Fig. 1C indicated by oval regions).

Bottom Line: However, siRNA accumulation was predominantly confined to ganglion cells layer as analysed 24 h post-injection.Furthermore, siRNA containing particles were localized along GFAP cytoskeleton of retinal astroglial cells hinting on intracellular localization of the siRNA CONCLUSIONS: In this work we demonstrated that siRNA can be efficiently delivered into the vertebrate retina in vivo with low-toxicity using a non-viral carrier, specifically Transit-TKO transfection reagent.Our report could raise a closer look on Transit-TKO transfection reagent as a promising siRNA carrier in vivo and be of interest for the researchers and companies who work on development of ocular RNAi techniques.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroanatomy and Molecular Brain Research, Ruhr-University Bochum, Germany. a.turchinovich@dkfz.de

ABSTRACT

Background: Gene silencing in the retina using RNA interference could open broad possibilities for functional studies of genes in vivo and for therapeutic interventions in eye disorders. Therefore, there is a considerable demand for protocols to deliver siRNA into the vertebrate retina. In this work we explored a possibility to deliver synthetic 21 bp siRNA into the mouse retina after intravitreal application using a non-viral carrier.

Methods: Fluorescently labelled synthetic 21 bp siRNA duplex was combined with Transit-TKO transfection reagent and injected intravitreally into adult mice eyes. Eyes cryostat sections and whole mount retinas were prepared 24-48 h post-injection, stained with either Hoechst 33342 (cell nuclei) or immunostained with anti-GFAP antibody (astroglia cells marker). Distribution of fluorescent siRNA signal in the retina was investigated.

Results: Single intravitreal injection of as little as 5 ng of siRNA combined with Transit-TKO transfection reagent by a modified protocol provided robust and non-toxic delivery of the siRNA into the retina. However, siRNA accumulation was predominantly confined to ganglion cells layer as analysed 24 h post-injection. Furthermore, siRNA containing particles were localized along GFAP cytoskeleton of retinal astroglial cells hinting on intracellular localization of the siRNA CONCLUSIONS: In this work we demonstrated that siRNA can be efficiently delivered into the vertebrate retina in vivo with low-toxicity using a non-viral carrier, specifically Transit-TKO transfection reagent. However, the capacity of siRNA delivered by our protocol to induce gene silencing in the retina has to be further evaluated. Our report could raise a closer look on Transit-TKO transfection reagent as a promising siRNA carrier in vivo and be of interest for the researchers and companies who work on development of ocular RNAi techniques.

Show MeSH
Related in: MedlinePlus