Gene transfer into retinal ganglion cells by in vivo electroporation: a new approach

Dezawa, Mari; Takano, Masahiko; Negishi, Hisanari; Mo, Xiaoliang; Oshitari, Toshiyuki; Sawada, Hajime

doi:10.1016/s0968-4328(01)00002-6

Cited by 79 publications

(54 citation statements)

References 15 publications

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“…For the mammalian central nervous system, this technique has been used to introduce genes into the fetal mouse brain, targeting the neural progenitor cells in the VZ (41)(42)(43). It has also been used for the retina in that Dezawa et al (44) recently reported the introduction of DNA into retinal GCs of the adult rat from the vitreous. However, at least in our experiments, we could see only a few GFP-positive cells when the GFP expression vector was injected into the vitreous of newborn rat eyes, followed by electroporation.…”

Section: Discussionmentioning

confidence: 99%

Electroporation and RNA interference in the rodent retina in vivo and in vitro

Matsuda

Cepko

2003

Proc. Natl. Acad. Sci. U.S.A.

956

848

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The large number of candidate genes made available by comprehensive genome analysis requires that relatively rapid techniques for the study of function be developed. Here, we report a rapid and convenient electroporation method for both gain-and loss-offunction studies in vivo and in vitro in the rodent retina. Plasmid DNA directly injected into the subretinal space of neonatal rodent pups was taken up by a significant fraction of exposed cells after several pulses of high voltage. With this technique, GFP expression vectors were efficiently transfected into retinal cells with little damage to the operated pups. Transfected GFP allowed clear visualization of cell morphologies, and the expression persisted for at least 50 days. DNA-based RNA interference vectors directed against two transcription factors important in photoreceptor development led to photoreceptor phenotypes similar to those of the corresponding knockout mice. Reporter constructs carrying retinal cell type-specific promoters were readily introduced into the retina in vivo, where they exhibited the appropriate expression patterns. Plasmid DNA was also efficiently transfected into retinal explants in vitro by high-voltage pulses.

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Section: Discussionmentioning

confidence: 99%

Electroporation and RNA interference in the rodent retina in vivo and in vitro

Matsuda

Cepko

2003

Proc. Natl. Acad. Sci. U.S.A.

956

848

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“…This method has been successfully used previously to transfect RGCs. [36][37][38][39] Although ELP does not target RGCs specifically, and transfected cells were distributed not only in the GCL, but also in the inner nuclear layer; most of GCL transfected cells were colocalized with RGCs. We found that cells in the nasal superior or nasal inferior quadrants of the retina were transfected more efficiently than cells in other retinal regions.…”

Section: Discussionmentioning

confidence: 99%

“…51,52 ELP-mediated gene delivery was performed as described previously with minor modifications. 37,39 In brief, animals were anesthetized with an intramuscular injection of 0.8 ml kg À1 of a cocktail containing 5 ml ketamine (100 mg ml À1 ), 2.5 ml xylazine (20 mg ml À1 ), 1.0 ml acepromazine (10 mg ml À1 ), and 1.5 ml normal saline. Four microliters of plasmid DNA (20 mg) was injected into the vitreous cavity with a 34-gauge needle 0.5 mm posterior to the limbus under stereoscopic microscopy.…”

Section: Gene Transfer Into Rgcs With Electroporationmentioning

confidence: 99%

Redox proteins thioredoxin 1 and thioredoxin 2 support retinal ganglion cell survival in experimental glaucoma

et al. 2008

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We investigated the neuroprotective effect of thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2) which play critical roles in the regulation of oxidative stress on retinal ganglion cells (RGCs) in a rat glaucoma model. Expression of Trx1 and Trx2 and Trx-interacting protein (Txnip) was observed in the RGC layer (GCL), nerve fiber layer and inner nuclear layer. Txnip-, Trx1-and Trx2-expressing cells in the GCL were primarily colocalized with RGCs. The increased Txnip protein level was observed 2 and 5 weeks after glaucoma induction. Trx1 level decreased 2 weeks after glaucoma induction and more prominently after 5 weeks. No change in Trx2 levels was detected. The effects of Trx1 and Trx2 overexpression on RGC survival were evaluated 5 weeks after glaucoma induction. In nontransfected and EGFP-transfected (used as a negative control) retinas, RGC loss was approximately 27% compared with control. The loss of RGCs in Trx1-and Trx2-transfected retinas was approximately 15 and 17%, respectively. Thus, Trx1 and Trx2 preserved 45 and 37% of cells, respectively that were destined to die in glaucomatous retinas. The results of this study provide evidence for the involvement of oxidative stress in RGC degeneration in experimental glaucoma and point to potential strategies to reduce its impact.

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“…MXIE control or MXIE-Chx10 vectors were injected into the retina of newborn mice, and DNA uptake was induced by electroporation (23,24). Retinas were harvested 21 days later, and GFP ϩ cell types were scored as above.…”

Section: Chx10 Drives Bipolar Cell Differentiation At the Expense Of mentioning

confidence: 99%

Chx10 is required to block photoreceptor differentiation but is dispensable for progenitor proliferation in the postnatal retina

Livne-Bar

Pacal

Cheung

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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In the Chx10-null ocular retardation (or J ) mouse, retinal progenitor cell (RPC) proliferation is impaired, and bipolar neurons, a late born cell type, fail to differentiate. It is unclear whether Chx10 is required to maintain proliferation throughout retinogenesis or whether the bipolar cell defect is an indirect effect of growth arrest. We show that Chx10 is dispensable for late-stage RPC proliferation but is essential to promote bipolar cell genesis in place of rods. Ectopic Chx10 expression drove bipolar instead of rod cell differentiation without affecting division. Converting Chx10 to an activator impaired bipolar cell differentiation, implying that repression is important for Chx10 activity. In the Chx10 null or J retina, only a small fraction of cells expressing mutated Chx10 mRNA were rods, but this fraction increased after p27 Kip1 inactivation, which partially rescues proliferation. Most significantly, acute Chx10 knockdown in the postnatal retina promoted rods in place of bipolar neurons without affecting division. Thus, Chx10 directly controls bipolar cell genesis by inhibiting rod differentiation independent of its temporally limited early effect on RPC proliferation.CVC domain ͉ homeobox ͉ homeodomain ͉ short-hairpin RNA

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Gene transfer into retinal ganglion cells by in vivo electroporation: a new approach

Cited by 79 publications

References 15 publications

Electroporation and RNA interference in the rodent retina in vivo and in vitro

Electroporation and RNA interference in the rodent retina in vivo and in vitro

Redox proteins thioredoxin 1 and thioredoxin 2 support retinal ganglion cell survival in experimental glaucoma

Chx10 is required to block photoreceptor differentiation but is dispensable for progenitor proliferation in the postnatal retina

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