1Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, University of Calgary, 2Department of Medical Genetics, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary
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Dixit, R., Lu, F., Cantrup, R., Gruenig, N., Langevin, L. M., Kurrasch, D. M., et al. Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation. J. Vis. Exp. (52), e2957, doi:10.3791/2957 (2011).
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.
3. Animal Preparation for Surgery
4. Incision and Exposure of Uterine Horns
5. Injection of DNA into Ventricles
7. Suture and Stapling
8. Post-surgical Care
9. Representative Results
The neocortex is the region of the CNS that is responsible for visual and sensorimotor processing and higher cognitive functioning. It is comprised of glutamatergic projection neurons and GABAergic interneurons that are derived from the dorsal and ventral telencephalon, respectively. To address the roles of different genes in neocortical development, we use in utero electroporation to either misexpress or block the expression (i.e., through the use of shRNA constructs) of different genes in the dorsal or ventral telencephalon1,3,4. To misexpress genes in the embryonic telencephalon, we use a bicistronic expression vector (pCIG2), containing a CMV-βactin promoter-enhancer and an internal ribosome entry site (IRES)-enhanced green fluorescent protein (EGFP; in pCIG2) cassette, allowing transfected cells to be detected by epifluorescence (Fig. 2A-C). We used this technology to introduce expression constructs into the telencephalon from E11.5 to E15.5 (data shown is at E12.5; Fig. 2). By allowing embryos to develop several days post-electroporation, we are able to track the differentiation and migration of GFP-labeled progenitors. As differentiation proceeds, 3 days post-electroporation GFP+ cells differentiate and migrate out of the ventricular zone (vz), subventricular zone (svz) and intermediate zone (iz) and into the cortical plate (cp; Fig. 2C). If brains electroporated at E12.5 are analyzed after 24 hr, we can monitor earlier events in progenitor maturation and neuronal migration by co-labeling with markers of apical progenitors (i.e., Pax6; Fig. 2A), basal progenitors (i.e., Tbr2; Fig. 2B) and postmitotic neocortical neurons (i.e., Tbr1; Fig. 2C).
The hypothalamus lies at the ventral base of the CNS and functions as a gateway between the endocrine and autonomic nervous systems. During development, the multiple nuclei that make up the mature hypothalamus differentiate from a common progenitor zone that lines the ventral-most region of the embryonic diencephalon. Currently, the molecular pathways that regulate progenitor cell proliferation, neuronal differentiation and the formation of hypothalamic nuclei are poorly understood. We used in utero electroporation to target reporter gene expression to the embryonic diencephalon by injecting DNA into the 3rd ventricle of E12 embryos.We then followed the distribution of GFP in differentiating neurons at E14. With this methodology, we have successfully targeted GFP expression to the thalamus, pre-thalamus and hypothalamus (Fig. 3A). Analysis of coronal sections reveals that we have successfully targeted GFP expression to progenitor cells that line the ventricular zone of the 3rd ventricle and neurons that migrate out to the mantle zone to form nuclei (Fig. 3B)
The retina is the neural layer of the eye, responsible for converting light photons into electrical impulses transmitted to the brain. It develops as an outpocketing of the embryonic diencephalon, and hence, is derived from the neural tube. The neural retina develops from an apical progenitor compartment that lies adjacent to the subretinal space, a cavity that separates the eye from the head mesenchyme. Targeting DNA constructs to retinal progenitors therefore requires that injections are made into a small target area. Using micromanipulators, we have managed to successfully target this small area in embryos from E13.5 to E15.5 (shown is E15.5 electroporations of pCIC with mCherry reporter; Fig. 4). If electroporated retinae are examined 24 hr post-transfection, we observe that most mCherry+ cells are located in the outer neuroblast layer (onbl), where dividing progenitors are located, and not in the retinal ganglion cell layer (gcl; inset image), where postmitotic cells localize.
Figure 1. Introduction to in utero electroporation set-up and methodology. (A) The key components of the surgical set-up include an Eppendorf Femtojet microinjector (1), Narishige micromanipulator with needle holder (2), Leica steromicroscope (3), fiber optic lighting system (4), ECM 830 Square Wave Electroporation System with electrodes (5) and heating pad (6). (A') The animal is anesthetized using a vaporizer for isoflurane anesthetic. (B,B') Briefly, after anesthesia, the uterine horns are exposed and DNA mixed with fast green is injected into the embryonic brain vesicles. A successful injection is visualized by tracking of the fast green dye (B'). (C,C') After the DNA is successfully injected, the paddle electrodes are placed on either side of the uterus, positioning the embryo so that the DNA will be pulled towards the positive pole into the brain region of choice. (D,D') Once all the desired embryos are injected, the uterus is pushed back into the abdomen (D), the peritoneum is sutured, and the skin is stapled (D'). The pregnant female is then allowed to recover and the electroporated pups are collected at the desired time points.
Figure 2. Representative example of dorsal telencephalic electroporation. (A-C) Photomicrograph of a coronal section through an E13.5 dorsal telencephalon electroporated at E12.5 with pCIG2. Electroporated GFP+ cells are analysed for their expression of markers of apical progenitors (Pax6+, red, A), basal progenitors (Tbr2+, red, B) and postmitotic neurons (Tbr1+, red, C). cp, cortical plate; svz, subventricular zone, vz; ventricular zone.
Figure 3. Representative example of diencephalic electroporation. (A) A ventral view of an E14 brain that has been electroporated at E12 with pCIG2. GFP epifluorescence is visible in the thalamus, pre- thalamus and hypothalamus, demonstrating the spread of the microinjected pCIG2 plasmid throughout the 3rd ventricle. (B) Photomicrograph of coronal section through electroporated brain, showing the migration of GFP+ cells out of the ventricular zone and into the mantle zone, where hypothalamic nuclei will form. 3V, third ventricle; T, thalamus; Hy, hypothalamus; mz, mantle zone; Tel, telencephalon; PreT, prethalamus; vz, ventricular zone.
Figure 4. Representative example of retinal electroporation. Coronal section through an E16.5 eye electroporated with pCIC at E15.5 showing the accumulation of mCherry+ cells in the outer neuroblast layer and not in Brn3b+ retinal ganglion cells in the ganglion cell layer. The inset is a higher magnification image of the boxed area. gcl, retinal ganglion cell layer; le, lens; onbl, outer neuroblast layer; re, retina.
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.
No conflicts of interest declared.
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.
|Fine scissors||Surgical Tools||Fine Science Tools||14078-10|
|Iris scissors, curved||Surgical Tools||Fine Science Tools||14061-10|
|Olsen-Hegar Ex-Delicate Needle Holder||Surgical Tools||Fine Science Tools||12002-12|
|Ring forceps, 9mm||Surgical Tools||Fine Science Tools||11103-09|
|Eye dressing Forcep||Surgical Tools||Fine Science Tools||11051-10|
|Dumont #7 DMX Forcep||Surgical Tools||Fine Science Tools||11271-30|
|Dumont #5 DMX Forcep||Surgical Tools||Fine Science Tools||11251-30|
|Tissue forcep-Adson||Surgical Tools||Fine Science Tools||11027-12|
|Reflex Clip Applier||Surgical Tools||World Precision Instruments, Inc.||500343|
|Perforated Spoon, 15 mm diameter||Surgical Tools||Fine Science Tools||10370-18|
|Autoclip Remover||Surgical Tools||Mikron||427637|
|Silk Black Braided Suture||Surgical Tools||Ethicon Inc.||K871|
|Reflex Skin Closure Stainless Steel Wound Clips||Surgical Tools||World Precision Instruments, Inc.||500346|
|ECM 830 Square Wave Electroporation System||Instruments||VWR international||58018-004|
|Tweezers w/Variable Gap 2 Round 5mm Platinum Plate Electrode||Instruments||Protech International, Inc.||CUY650P5|
|Tweezers w/Variable Gap 2 Round 7mm Platinum Plate Electrode||Instruments||Protech International, Inc.||CUY650P7|
|Eppendorf Femtojet Microinjector||Instruments||VWR international||CA62111-488|
|Foot Control for Eppendorf Femtojet Microinjector||Instruments||VWR international||CAACCESS (misc.)|
|Bransonic Ultrasonic Cleaner Model 1510R-DTH||Instruments||VWR international||CA33995-534CPN-952-118|
|Sutter P97 Micropipet Puller||Instruments||Sutter Instrument Co.||P-97|
|Micropipettes - Borosilicate with filament O.D.: 1mm, I.D.: 0.78 mm, 10 cm length||Instruments||Sutter Instrument Co.||BF100-78-10|
|3-Axis Coarse Manipulator||Instruments||Carl Zeiss, Inc.||M-152|
|Magnetic Holding Device for micromanipulator||Instruments||World Precision Instruments, Inc.||M1|
|Steel Base Plate for micromanipulator||Instruments||World Precision Instruments, Inc.||5052|
|Micropipette Holder||Instruments||World Precision Instruments, Inc.||MPH3|
|Micropipette Handle||Instruments||World Precision Instruments, Inc.||5444|
|Vaporizer for isoflurane anesthetic||Instruments||Porter Instruments Company||MODEL 100-F|
|Metriclean2 Low foaming solution for sonicating surgical tools||Surgical Reagents||Metrex Research Corporation||10-8100|
|Gentamicin 40mg/ml in 0.2 g methylene blue antibiotic spray after suturing||Surgical Reagents||Sigma-Aldrich||G1264|
|Germex for sterilizing surgical tools||Surgical Reagents||Vétoquinol||DIN# 00141569|
|BNP ophthalmic ointment||Surgical Reagents||Vétoquinol||DIN# 00516414|
|NairÃ®||Surgical Reagents||Church & Dwight Co.||commercially available|
|Stanhexidine 4% w/v skin cleaner||Surgical Reagents||Omega Laboratories Inc.||01938983|
|Buprenorphine (Temgesic) analgesic||Surgical Reagents||Merck & Co.||531-535|
|Sulpha Ã¢â¬Å25Ã¢â¬Â sulphamethazine oral antibiotic||Surgical Reagents||Professional Veterinary Laboratories||DIN# 00308218|
|Lactated Ringer Solution||Surgical Reagents||Baxter Internationl Inc.||DIN# 0061085|
|Saline - 0.9% sodium chloride||Surgical Reagents||B. Braun Medical||DIN# 01924303|
|Inhalation Anesthetic - Isoflurane USP||Surgical Reagents||Pharmaceutical Partners of Canada||DIN# 02237518|
|Fast Green FCF||Surgical Reagents||Sigma-Aldrich||F7252|