NEPA21_Retina_EP

CUY701P2E / CUY701P2L

CUY675P5

CUY650P7

A small hole was made with a 31-gauge needle 0.5 to 1.0 mm
posterior to the limbus. The tip of the needle was viewed as it entered the vitreous at a 40° to 50° angle through the dilated pupil. 10 μg plasmid DNA in 4 μL TE buffer was injected into the vitreous.

The eye was then immediately grasped between tweezerz-type electrodes, with the cathode attached to the corneal electrode and the anode with the slit attached to the scleral electrode.

(A) Tweezer-type electrodes were placed on the corneal surface of either eye of a 1-month-old Sprague-Dawley rat.

(B) The current was applied with the positive electrode contralateral to the injected eye. After prior injection of plasmid DNA into the subretinal space of the right eye, this arrangement electrophoresed the negatively-charged DNA toward the RPE layer (arrowheads).

Target

Publication

Electrode

Protocol

.

SponGee: a genetic tool for subcellular and cell-specific CGMP manipulation

Cell Rep. 2019 Jun 25;27(13):4003-4012.e6. doi: 10.1016/j.celrep.2019.05.102.

Gene Transfer of Prolyl Hydroxylase Domain 2 Inhibits Hypoxia-inducible Angiogenesis in a Model of Choroidal Neovascularization.

Quantitative Analysis of the ThrbCRM1-centered Gene Regulatory Network

Biol Open,8 (4) 2019 Apr 26

Identification of a Retina-Specific Otx2 Enhancer Element Active in Immature Developing Photorecetpors

Dev Biol, 360 (1), 241-55 2011 Dec 1

CUY700P3L

Embryos

in ovo

Lineage Tracing Analysis of Cone Photoreceptor Associated Cis-Regulatory Elements in the Developing Chicken Retina

Sci Rep, 9 (1), 9358 2019 June 27

Single Cell profiling of CRISPR/Cas9-induced OTX2 deficient retinas reveals fate switch from restricted progrnitors

bioRxiv. 2019 Februarys 2, https://dio.org/10.1101/538710

Irx4-mediated regulation of Slit1 expression contributes to the definition of early axonal paths inside the retina

Development, Volume 130, Issue 6, Pages 1037-1048, March 2003

CUY614 & CUY615

Single Cell profiling of CRISPR/Cas9-induced OTX2 deficient retinas reveals fate switch from restricted progrnitors

bioRxiv. 2019 Februarys 2, https://dio.org/10.1101/538710

ex vivo

Lineage Tracing Analysis of Cone Photoreceptor Associated Cis-Regulatory Elements in the Developing Chicken Retina

Sci Rep, 9 (1), 9358 2019 June 27

Retina

In vivo / ex vivo

Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina

Cell and Tissue Research, Volume 332, Number 2, Pages 195-209, May 2008

CUY701P2E

Inhibition of Müller glial cell division blocks regeneration of the light-damaged zebrafish retina

Developmental Neurobiology, Volume 68, Issue 3, Pages 392-408, 15 February 2008

With respect to gene delivery into Mouse Embryo Eye/Retina (and Brain) one can use the tungsten needle type electrode. However, as the mouse embryo is very small it is often hard to inject DNA into the embryo’s eye.

Some clients have reported using  the CUY675P5 electrode

It is structurally possible to use this electrode for both Embryo (in utero) and Mouse Eye electroporation.

(Please note the table below).

	CUY675P5	..		CUY650P7
The left eye and the optic nerve were exposed.  A small hole was made with a 31-gauge needle 0.5 to 1.0 mm posterior to the limbus. The tip of the needle was viewed as it entered the vitreous at a 40° to 50° angle through the dilated pupil. 10 μg plasmid DNA in 4 μL TE buffer was injected into the vitreous. The eye was then immediately grasped between tweezerz-type electrodes, with the cathode attached to the corneal electrode and the anode with the slit attached to the scleral electrode.			Arrangement of electrodes for in vivo electroporation for RPE transfection. (A) Tweezer-type electrodes were placed on the corneal surface of either eye of a 1-month-old Sprague-Dawley rat. (B) The current was applied with the positive electrode contralateral to the injected eye. After prior injection of plasmid DNA into the subretinal space of the right eye, this arrangement electrophoresed the negatively-charged DNA toward the RPE layer (arrowheads).

As one can see, however, there is a cut-out on one side of the CUY675P type electrodes.  So, for this application we are not convinced that it is the optimum electrode for the application.

Rather, our recommendation is for the CUY650P7 electrode.  Note the attached CUY650P7 Technical Drawing.

This is the electrode most of our clients choose.  It has a fully rounded set of electrode paddles and therefore covers a larger area of the target.  In addition, this electrode has a much wider application range and can be used, for example, for or (whole) Cultured Mouse Embryos and Large Tissue EP.

Dr. Matsuda uses the CUY650P7 electrode in his articles contained in the ‘EP with Retina’ below).

Takahiko Matsuda and Constance L. Cepko

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

PNAS, Volume 101, Number 1, Pages 16-22, 6 January 2004

CUY650P5 technical Drawing

–          CUY671P1 or                                        Technical Drawing: CUY671P1

–          CUY670 Technical Drawing: CUY670

Protocol Recommendation:

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

–  ‘Protocol for in vivo electroporation into mouse/rat retina’

–  ‘Compensation by tumor suppressor genes during retinal development in mice and humans‘

For your further information, note the article titled:

–  ‘A role for ligand-gated ion channels in rod photoreceptor development’.

In this article and ‘Electroporation and RNA interference in the rodent retina in vivo and in vitro’ above genes are transfected into mouse and rat retina.  We believe that the method outlined in these articles can be applied to Gene Therapy