В естественных условиях Электропорация развивающихся Мышь Retina
Summary
Метод включения плазмидной ДНК в мышиных клеток сетчатки для целей осуществления любой выигрыш или потеря функции исследования<em> В естественных условиях</em> Представлена. Этот метод капитализирует на кратковременное повышение проницаемости клеточных мембран плазмы индуцированных применением внешнего электрического поля.
Abstract
The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10.
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate.
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.
Protocol
Discussion
В естественных условиях электропорации представляет быстрый и эффективный метод для преобразования клеток сетчатки с помощью плазмидов экспрессии ДНК. Этот метод позволяет экспериментатору выполнить усиление функции исследования эктопически введения гена под контролем повсе…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Эта работа финансировалась NIH R01EY020560-01 и WM Keck молодой ученый в области медицинской премии Research. Авторы хотели бы поблагодарить Иосифа Bedont за его помощь во время визуализации сетчатки препаратов и инъекций.
Materials
| Name of Reagent | Company | Catalogue Number | Comments |
|---|---|---|---|
| Buffer Saturated Phenol | Invitrogen | 15513-039 | N/A |
| Chloroform | J.T. Baker | 9180-03 | N/A |
| Sodium Acetate | J.T. Baker | 3470-05 | 3 M stock |
| Fast Green FCF | Fisher Biotech | BP123-10 | 10 % stock |
| Isopropyl alcohol prep | Tyco Healthcare | 6918 | N/A |
| 30-guage needle | Terumo Medical Corp. | SG2-3013 | N/A |
| Exmire microsyringe | Ito Corporation | MS*E05 | N/A |
| Tweezertrode (tweezer electrode) | BTX Instrument, Genetronics Inc. | 522 | N/A |
| Electro Square Porator (electroporator) | BTX Instrument, Genetronics Inc. | ECM 830 | N/A |
| O.C.T. Compound | Sakura Finetek USA | 4583 | N/A |
References
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