Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Written By India Gate on Tuesday, June 28, 2011 | 1:02 AM

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.

Discussion

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. Finally, targeted overexpression or misexpression of key genes in both wild type and/or genetically mutant mouse lines can be used to study cell fate decisions. The high throughput of this assay is critical as it allows for the testing of many combinations of factors in a very short time. One note of caution is that this procedure does cause changes in gene expression in cells that line the needle entry site (i.e., injury-response genes upregulated in wound; as observed by us and others13). It is thus recommended to focus on electroporated cells outside of the wound site. In addition, embryonic survival rates are low when first learning this technique but quickly rise to >95% with practice. To date, we have successfully used in utero electroporation technologies to identify genes regulated by proneural bHLH transcription factors in the telencephalon4. We have also validated the use of this technique for the analysis of telencephalic cis-regulatory elements3.

Disclosures

No conflicts of interest declared.

Acknowledgements

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.

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. http://www.jove.com/details.php?id=2957 doi: 10.3791/2957. J Vis Exp. 52 (2011).