Summary

Эффективное доставки генов в нескольких территориях ЦНС Использование In Utero Электропорация

Published: June 23, 2011
doi:

Summary

В электропорации внутриутробно позволяет для быстрой доставки гена в пространственно-временном и-контролируемым образом в развивающихся центральной нервной системы (ЦНС). Здесь мы опишем хорошо адаптируется внутриутробно электропорации протокола, которые могут быть использованы для доставки выражение конструкции на несколько эмбриональных областях ЦНС, в том числе конечного мозга, промежуточного мозга и сетчатки.

Abstract

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.

To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Protocol

1. Настройка Настройка хирургических область, как показано на рис. 1А, '. Ключевые компоненты настройки включают Эппендорф Femtojet microinjector, Narishige микроманипулятора с иглодержателя, Leica steromicroscope, волоконно-оптические системы освещения, ECM 830 квадратных системы Электропорация волны с э?…

Discussion

Внутриутробное электропорации могут быть использованы для анализа разнообразных процессов развития. Например, трансфекции репортер генов, таких как GFP, mCherry или щелочной фосфатазы может быть использован для проведения линии отслеживания и нейронных экспериментов миграции. Кром?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Авторы хотели бы поблагодарить Ева Hadzimova, Пьер Маттар и Кристофер Ковач для их первоначальной работы по созданию внутриутробно электропорации технологии в CS лаборатории. Эта работа финансировалась Канадский институт исследований в области здравоохранения (CIHR) грант (44 094 СС) и CIHR / Фонд борьбы слепоты (FFB) новые команды Грант (00933-000) для CS и Альберта детской больницы Фонд исследований Грант ДМК. РД была поддержана Надежда стипендии CIHR Канаде, RC поддерживается Студенчество FFB и LML был поддержан грант CIHR Обучение генетики и развития ребенка.

Materials

Name of reagent Company Catalogue Number Category
Fine scissors Fine Science Tools Inc. 14078-10 Surgical Tools
Iris scissors, curved Fine Science Tools Inc. 14061-10 Surgical Tools
Olsen-Hegar Ex-Delicate Needle Holder Fine Science Tools Inc. 12002-12 Surgical Tools
Ring forceps, 9mm Fine Science Tools Inc. 11103-09 Surgical Tools
Eye dressing Forcep Fine Science Tools Inc. 11051-10 Surgical Tools
Dumont #7 DMX Forcep Fine Science Tools Inc. 11271-30 Surgical Tools
Dumont #5 DMX Forcep Fine Science Tools Inc. 11251-30 Surgical Tools
Tissue forcep-Adson Fine Science Tools Inc. 11027-12 Surgical Tools
Reflex Clip Applier World Precision Instrument 500343 Surgical Tools
Perforated Spoon, 15 mm diameter Fine Science Tools Inc. 10370-18 Surgical Tools
Autoclip Remover Mikron 427637 Surgical Tools
Silk Black Braided Suture Ethicon Inc. K871 Surgical Tools
Reflex Skin Closure Stainless Steel Wound Clips World Precision Instruments 500346 Surgical Tools
ECM 830 Square Wave Electroporation System VWR-CanLab 58018-004 Instruments
Tweezers w/Variable Gap 2 Round 5mm Platinum Plate Electrode Protech International Inc. CUY650P5 Instruments
Tweezers w/Variable Gap 2 Round 7mm Platinum Plate Electrode Protech International Inc. CUY650P7 Instruments
Eppendorf Femtojet Microinjector VWR CanLab CA62111-488 Instruments
Foot Control for Eppendorf Femtojet Microinjector VWR CanLab CAACCESS (misc.) Instruments
Bransonic Ultrasonic Cleaner Model 1510R-DTH VWR CanLab CA33995-534 CPN-952-118 Instruments
Sutter P97 Micropipet Puller Sutter Instrument, Carsen Group Inc. P-97 Instruments
Micropipettes – Borosilicate with filament O.D.: 1mm, I.D.: 0.78 mm, 10 cm length Sutter Instrument BF100-78-10 Instruments
3-Axis Coarse Manipulator Carl Zeiss Canada Inc. M-152 Instruments
Magnetic Holding Device for micromanipulator World Precision Instruments M1 Instruments
Steel Base Plate for micromanipulator World Precision Instruments 5052 Instruments
Micropipette Holder World Precision Instruments MPH3 Instruments
Micropipette Handle World Precision Instruments 5444 Instruments
Stereomicroscope Leica MZ6 Instruments
Vaporizer for isoflurane anesthetic Porter Instruments Company MODEL 100-F Instruments
Metriclean2 Low foaming solution for sonicating surgical tools Metrex Research Corporation 10-8100 Surgical Reagents
Gentamicin 40mg/ml in 0.2 g methylene blue antibiotic spray after suturing Sigma Aldrich G1264 Surgical Reagents
Germex for sterilizing surgical tools Vétoquinol DIN# 00141569 Surgical Reagents
BNP ophthalmic ointment Vétoquinol DIN# 00516414 Surgical Reagents
Nair® Distributed by Church & Dwight Co., Inc. commercially available Surgical Reagents
Stanhexidine 4% w/v skin cleaner Omega Laboratories Inc. 01938983 Surgical Reagents
Buprenorphine (Temgesic) analgesic Schering-Plough 531-535 Surgical Reagents
Sulpha “25” sulphamethazine oral antibiotic Professional Veterinary Laboratories DIN# 00308218 Surgical Reagents
Lactated Ringer Solution Baxter Corporation DIN# 0061085 Surgical Reagents
Saline – 0.9% sodium chloride B-Braun Medical Inc. DIN# 01924303 Surgical Reagents
Inhalation Anesthetic – Isoflurane USP Pharmaceutical Partners of Canada Inc. DIN# 02237518 Surgical Reagents
Fast Green FCF Sigma-Aldrich F7252 Surgical Reagents

References

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Cite This Article
Dixit, R., Lu, F., Cantrup, R., Gruenig, N., Langevin, L. M., Kurrasch, D. M., Schuurmans, C. Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation. J. Vis. Exp. (52), e2957, doi:10.3791/2957 (2011).

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