In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.
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.
In utero electroporation can be used to analyze a wide variety of developmental processes. For example, transfection of reporter genes such as GFP, mCherry or alkaline phosphatase can be used to conduct lineage tracing and neuronal migration experiments. Alternatively, Cre recombinase can be transiently expressed to selectively eliminate a floxed allele in a spatially- and/or temporally-controlled manner. Furthermore, shRNA or dominant negative constructs can be electroporated to knockdown target gene function. …
The authors have nothing to disclose.
The authors would like to thank Eva Hadzimova, Pierre Mattar and Christopher Kovach for their initial work in establishing in utero electroporation technology in the CS lab. This work was funded by a Canadian Institute of Health Research (CIHR) grant (MOP 44094) and CIHR/Foundation Fighting Blindness (FFB) Emerging Team Grant (00933-000) to CS and an Alberta Children’s Hospital Research Foundation Grant to DMK. RD was supported by a CIHR Canada Hope Scholarship, RC is supported by an FFB Studentship and LML was supported by a CIHR Training Grant in Genetics and Child Development.
Name of reagent | Company | Catalogue Number | Category |
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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 |