This work was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft).

1. Preparation of DNA and Glass Micropipettes for Injection
<ol>
	<li>Good quality glass micropipettes are essential to reduce initial high abortion rate due to loss of amniotic fluid. The procedure to pull glass micropipettes has been well documented13,18,25. Use 1.2 mm diameter capillaries pulled in a conventional Sutter P-97 device with the settings P=500; Heat=300; Pull=40; Velocity=50; Time=50. Fit the puller with 3 mm "trough" filaments (Sutter Instrument FT330B). The 2 mm size filaments have yielded for us less satisfactory results. On the other hand, beveling of the micropipette tips does not seem to improve results for us.</li>
	<li>Dissolve purified, endotoxin-free plasmid DNA in PBS (Cell Culture Grade) containing 0.1% Fast Green to a final concentration of 1 to 2 &mu;g/&mu;l. The Fast Green will make the injected solution visible in the embryonic brain ventricle.</li>
	<li>Load 10 &mu;l of the DNA solution into the glass micropipette.</li>
	<li>Connect the glass micropipette to the injection system (Pico Pump) or mouth pipette.</li>
</ol>
2. Anesthesia
Prepare the surgical table with a heating pad and the surgical instruments. Turn on the cold-light sources to facilitate the visualization of the embryos. Disinfect the surgical tools using for instance a glass bead sterilizer.
Three different anesthesia procedures are possible for this protocol (see Discussion). Here we will describe the one we consider the best, using isoflurane inhalation for anesthesia induction (flow rate 0.5 L/min) as well as maintenance (flow rate 1 L/min).
<ol>
	<li>Introduce the pregnant mouse in a small transparent (so the mouse remains visible) container connected to the Komesaroff Mark 5 Anesthetic Device by a short length of tubing.</li>
	<li>Fill the vaporizer with isoflurane, then open the oxygen bottle attached to the device. Blow one or two pulses of isoflurane/oxygen (0.5 L/min) into the container holding the mouse and watch for narcosis to set in.</li>
	<li>Take the mouse out, cover its eyes with ointment to prevent them from drying and fit the flow anesthesia mask on its head immediately.</li>
</ol>
3. Laparotomy
<ol>
	<li>Place the mouse belly up on the heating pad (otherwise its body temperature will go down very fast, decreasing chances of recovery) and secure its body into place by fixing its four legs to the sides with tape. Assess the depth of anesthesia by checking for loss of response to reflex stimulation (e.g. toe or tail pinch with firm pressure).</li>
	<li>Shave the abdominal surface and disinfect it with the iodophorpovidone-iodine (Braunoderm).</li>
	<li>Make a longitudinal incision (1 to 1.5 cm long) on the abdominal skin. Then cut the peritoneum. Place cotton gauze around the incision. Make one uterine horn visible and pull it out carefully with blunt forceps onto the PBS-rinsed gauze. Rinse the uterus with PBS very often to keep it always moistened (Figures 2A and 2B). During the entire procedure avoid pulling the mesometrium or the uterus tight, since high pressure inside the uterus will transmit to the embryo increasing the chances of loss of fluid upon injection resulting in abortion.</li>
</ol>
4. DNA Injection into the Brain Ventricle
<ol>
	<li>Hold the uterus in such a way that the brain ventricles can be visualized. Do not extensively reposition an embryo that lies in an unfavorable position - this only increases the chances of abortion.</li>
	<li>Looking at the embryo's head from top, localize visually the gap or fissure between the left and right cortical hemispheres. The hemispheres are easy to distinguish and the lateral ventricles inside them (not to be targeted) can usually be perceived as somewhat darker shapes. If an embryo is found to be perfectly oriented for DNA injection (Figures 2C and 2D), it is possible to pierce the uterine wall and enter the third ventricle at once. Hold the glass micropipette at 45&deg; to the uterine wall and puncture it at the rostral end of the gap between cortical hemispheres, penetrating for about 1 mm. In this way the tip of the glass micropipette will enter the third ventricle of the brain (not the lateral ventricle) (Figures 2C and 2D). In embryos less favorably oriented it is useful to first pierce the uterine wall (always at 45&deg;) in the vicinity of the embryonic head, place the micropipette tip in the right position between the cortical hemispheres and only then perforate the brain. Inject about 1 &mu;l of DNA solution into the third ventricle (a good injection fills the ventricle with green fluid). Repeat the same procedure with all embryos of one uterus horn. This allows some time for the DNA solution to mix evenly with the ventricular fluid and reach the entire neuroepithelium.</li>
</ol>
5. Targeting the Hypothalamus for Electroporation
<ol>
	<li>Switch the electroporator on and adjust settings according to the embryonic age (for E12.5 we use 5 square-wave pulses, 50 V, 50 msec ON/950 msec OFF). Use as positive pole the stainless steel needle electrode (CUY550-10) and as negative pole a round flat electrode (CUY700P4L). (It is important that the electrodes are smaller than the embryo, since otherwise the current just flows around the embryo but not through it.)</li>
	<li>Select for electroporation those embryos whose dorsal side is up (i.e. turned towards the experimenter) (as most of them are), and discard those whose orientation is not favorable. It is now safe to touch the uterus with ethanol-disinfected fingers or with gloves. Holding the uterus with forceps, however, would interfere with the flow of current during electroporation.</li>
	<li>Pierce the uterine wall by the embryo's head between the amniotic sac and the placenta by thrusting downwards through it with the tip of the needle electrode. Use the index finger of the other hand as thrust block. About 5 mm of the electrode tip must be now between the amniotic sac and the uterine wall, at about the level of the midbrain (Figures 2E and 2F). Remember that this is the "targeting electrode", so its position will determine which part of the hypothalamic neuroepithelium is most likely to get transfected. The embryo has to be very gently "squeezed" between the electrodes.</li>
	<li>With the other hand, position the round flat electrode outside of the uterus wall on the opposite side of the embryo's head (Figures 2E and 2G).</li>
	<li>Use the pedal switch to apply voltage (50 V, 50 msec ON, 950 msec OFF, 5 square-wave pulses).</li>
	<li>Slowly pull the needle electrode out of the uterus while holding back the uterus with the index finger of the other hand - if amniotic liquid is lost through the punctured wall, usually the embryo will undergo abortion. Repeat the procedure with all the embryos.</li>
	<li>Return the uterus horn into the abdomen and repeat the injection and electroporation in the remnant horn.</li>
</ol>
6. Finishing the Surgery
<ol>
	<li>After injection and electroporation of all embryos, place the uterus back into the abdomen very carefully, positioned exactly as they were before and moist the peritoneal cavity with saline before closing it.</li>
	<li>Suture the peritoneum with surgical catgut (Vicryl Polyglactin 910, 5-0, Ethicon V4914). For the closure of the skin use "interrupted stitch" methodwith a more resistant suture (Supramid Nylon, 6-0, Serag Wiessner TO 07171L).</li>
	<li>Disinfect the abdomen surface with povidone-iodine(Braunoderm) and inject a non-steroid anti-inflammatory subcutaneously (e.g. 100 &mu;l of a 1:10 solution of Rimadyl in 0.9% NaCl) or, even better, an opioid like buprenorphine (Temgesic, 0.05 to 0.1 mg/kg body weight in 0.9% NaCl)to relieve pain.</li>
	<li>Remove the mother from the anesthetic machine and place it in a cage which is heated by a heating pad. Monitor the mouse continually until it is completely recovered from the anesthesia. Later on, check the animals daily to insure they are recovering from the procedure without any sign of infection or pain.</li>
</ol>
7. Analyzing the Results
<ol>
	<li>Harvest the embryos (or postnatals) according to the desired day of analysis. Postnatal mice (1 to 2 day old) are killed by decapitation.</li>
	<li>Separate every embryo according to the previous electroporation annotation. Dissect the brain under a stereomicroscope and check under the microscope for green fluorescent signal (from the GFP reporter) in the appropriate region.</li>
	<li>The selected brain can be analyzed "fresh": fix the tissue for a short time in 4% paraformaldehyde and embed in agarose 4% or in gelatin-albumin, then section on a vibratome-type device (Figure 3). For more detailed immunohistochemical analysis (Figure 4), cryo-protect the brain in 30% saccharose solution, embed in OCT mounting medium (Tissue Tek) then cut 20 &mu;m sections in a cryostat.</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+promoters+for+driving+efficient+transgene+expression+in+neonatal+porcine+islets.">Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets.</a> Xenotransplantation. 14, 119-125 (2007).</li></ol>

The authors declare that they have no competing financial interests.

About the anesthesia: Since in utero electroporation into the hypothalamus can be technically arduous and require longer narcosis times, we prefer to induce and maintain anesthesia through administration of a mixture of oxygen and isoflurane. In our experience, animals can remain suitably anaesthetized in this way for periods of up to one hour at least, the recovery of the mother is very fast, and embryo survival improved. Other approaches to anesthesia are also available. The most simple procedure consis...

Genetic manipulation of the embryonic mouse brain is a preferred approach to learn about developmental regulation. The generation of mutant mouse lines however is slow and expensive. One powerful method to introduce specific genetic changes in developing neurons of the mammalian brain is in utero electroporation. Essentially, the technique consists of transfecting DNA into the embryonic brain neuroepithelium by means of electric pulses, then allowing the embryo to survive for a certain period of time, collect the brain and examine them for possible novel, informative phenotypes. In this way, the experimenter can test hypotheses almost immediately without the long waiting periods necessary for the production of mouse mutants.	
	Transfection of DNA into developing embryos started with in ovo electroporation on chick embryos1. The essential proof-of-concept for the mouse was performed in culture2. This was soon followed by the first descriptions of the technique on the mouse in utero3,4.	
	The main problem is to transfect the brain of embryos developing in utero without killing them or the mother. Learning to perform the necessary surgery (laparotomy, injection, electroporation) requires a long training period. Once the surgery has been mastered to the point where the embryo survival ratio is acceptable, the next key question is: which brain structures are accessible? Not surprisingly, the first published papers showing results obtained with in utero electroporation focused on cortical development5-9. This is still true for most of the publications using this technique, since the region of the developing mouse brain most accessible to surgical procedures is the cortex (Figure 1). The procedure for in utero electroporation into the cortex has been described in print10 and in video11-14. A modification of the technique can be used to target a ventral part of the telencephalon, the basal ganglia15.	
	Beyond the telencephalon, the diencephalon (classically divided into thalamus and hypothalamus) is a region of the forebrain more difficult to reach. A small number of papers reports targeting of its dorsal and most accessible part, the thalamus16-19.	
	The hypothalamus is the most ventral part of the forebrain, therefore the one localized most deeply from the dorsal surface (cortex) (Figure 1). This region remains a difficult challenge for researchers committed to genetically manipulate the mouse brain in utero. To our knowledge, only very few articles report on results of in utero transfection into the mouse hypothalamus 20,21. However, the functional importance of the hypothalamus cannot be overstated, since it regulates behaviors like eating and drinking, mating, breeding and parenting22. Moreover, alterations in hypothalamic development contribute to originate later in life conditions like obesity, hypertension, diabetes and precocious puberty23. Being able to alter genetically the hypothalamus during development would provide a very powerful tool to understand it.	
	The basic surgical protocol for the laparotomy of pregnant mice that we use here is similar to that used in other protocols11,13,14,24. We will describe them here briefly for completeness. Key to our procedure, on the other hand, are the type of anesthesia, the place of injection, the type of electrodes and the insertion and position of the positive electrode with respect to the embryo's head. We prefer to induce and maintain anesthesia through gas inhalation over simple intraperitoneal anesthesia, since the former allows for the somewhat longer periods of narcosis required for a difficult surgery. Isoflurane inhalation results in faster recovery from anesthesia, since usually the mother demonstrates normal behavior already minutes after the surgery. The easiest point of injection of the DNA solution with the glass micropipette is the lateral ventricle, which however is completely unsuitable for hypothalamus electroporation. Injection directly in the third ventricle is indeed crucial to target deep diencephalic structures. It is possible to transfect the hypothalamus from E12.0 or E12.5 with standard, off-the-shelf electrodes. We have found some of the electrodes manufactured by Nepa Gene (Chiba, Japan) particularly suited to this purpose.	
	With our procedure we obtain transfection of the entire hypothalamic neuroepithelium or partial, regional transfection depending on electrode orientation. Here we demonstrate the technique by transfecting the mammillary body, arguably the deepest and most recessed of all hypothalamic nuclei. Additionally, we show detailed histological analysis of the transfected cells down to the cellular level of resolution.	
	A comparison of in utero electroporation with other approaches to transfecting the mouse developing brain in utero can be found in the Discussion section.

<table><tbody><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Acepromazine</td><td>Sanofi GmbH</td><td></td><td>anesthetic</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>HDG9623</td><td>anesthetic</td></tr><tr><td>Ketamin</td><td>Pharma GmbH</td><td></td><td>anesthetic</td></tr><tr><td>Fast Green</td><td>Fluka</td><td>44715</td><td>&amp;nbsp;</td></tr><tr><td>Rimadyl</td><td>Pfizer</td><td></td><td>non-steroidal anti-inflammatory</td></tr><tr><td>Braunoderm</td><td>Braun</td><td>3887138</td><td>povidone-iodine</td></tr><tr><td>Phosphate Buffer Saline PBS</td><td>Gibco</td><td>14190</td><td>&amp;nbsp;</td></tr><tr><td>Temgesic (buprenorphine)</td><td>Essex Pharma</td><td></td><td>opioid analgesic</td></tr><tr><td>Eye Ointment</td><td>Pan-Ophtal</td><td>7136926</td><td>&amp;nbsp;</td></tr><tr><td>Xylazine</td><td>Bayer</td><td></td><td>&amp;nbsp;</td></tr><tr><td>&amp;nbsp;</td><td></td><td></td><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Anaesthetic Device Komesaroff Mark-5</td><td>Medical Developments Australia</td><td>ACN 004 903 682</td><td>&amp;nbsp;</td></tr><tr><td>Capillary puller P-97</td><td>Sutter Instrument Co.</td><td>P-97</td><td>&amp;nbsp;</td></tr><tr><td>Compresstome</td><td>Precisionary Instr.</td><td>VF-300</td><td>Vibratome-type device</td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM700</td><td>&amp;nbsp;</td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050S</td><td>&amp;nbsp;</td></tr><tr><td>Electroporator</td><td>Nepa Gene Co. Ltd.</td><td>CUY21EDIT</td><td>&amp;nbsp;</td></tr><tr><td>Electrode 1</td><td>Nepa Gene Co. Ltd.</td><td>CUY550-10</td><td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td></tr><tr><td>Electrode 2</td><td>Nepa Gene Co. Ltd.</td><td>CUY700P4L</td><td>Cover Round Platinum Plate 4 mm diameter</td></tr><tr><td>Fiberoptic cold light source</td><td>Leica</td><td>KL2500 LCD</td><td>&amp;nbsp;</td></tr><tr><td>Glass capillaries</td><td>Harvard Apparatus</td><td>GC120T-15</td><td>1. 2 mm O.D. x 0. 94 mm I.D.</td></tr><tr><td>Glass bead sterilizer</td><td>Fine Science Tools</td><td>FST250</td><td>&amp;nbsp;</td></tr><tr><td>Heating pad</td><td>Harvard Apparatus</td><td>py872-5272</td><td>&amp;nbsp;</td></tr><tr><td>Injection device</td><td>World Precision Instruments</td><td>Pneumatic Pico Pump PV820</td><td>&amp;nbsp;</td></tr><tr><td>Suture Thread Coated Vicryl</td><td>Ethicon</td><td>V4914</td><td>Peritoneal Suture</td></tr><tr><td>Suture Thr. Supramid</td><td>Serag Wiessner</td><td>TO07171L</td><td>Skin Suture</td></tr></tbody></table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Most hypothalamus neurons are born between E11.5 to E15.2, according to birth-dating analysis in the rat26 translated into the somewhat shorter mouse development27,28. The peak of hypothalamic neurogenesis is reached at E12.529-31. Accordingly, at the transfection age chosen for the present study (E12.5), a large proportion of hypothalamic neurons can be labeled at any given rostro-caudal level.
Analysis at E18.5 on thick vibratome-type sections (Figur...

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

genetic-manipulation-mouse-developing-hypothalamus-through-utero

Research

JoVE Journal

Neuroscience

15.8K Views.  University of Heidelberg. Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Video: Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Developmental Biology

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biology

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle (reviewed in 1). The rhombic lip can be subdivided into the upper rhombic lip (URL) which encompasses rhombomere 1 (r1) and generates neurons of the cerebellum and the lower rhombic lip (LRL) which gives rise to diverse neuronal brainstem lineages 2-4. LRL derivatives include the auditory neurons of the cochlear nuclei and those of the precerebellar nuclei that are involved in regulating balance and motor control 5-8. Neurogenesis from the LRL occurs over a large temporal window that encompasses embryonic days (E) 9.5-16.55, 9. Different neuronal lineages emerge from the LRL as postmitotic cells (or are born) during distinct developmental days during this neurogenic window. 
Electroporation of gene expression constructs can be used to manipulate gene expression in LRL progenitors and can potentially change the fate of the neurons produced from this region 10-12. Altering gene expression of LRL progenitors in the mouse via in utero electroporation has been highly successful for manipulating lineages born on embryonic day E12.5 or later 10, 12-14. In utero electroporations prior to E12.5 have been unsuccessful primarily due to the lethality associated with puncturing the fourth ventricle roofplate, a necessary step in delivering exogenous DNA that is electroporated into the LRL. However, many LRL derived lineages arise from the LRL earlier than E12.5 9. These earlier born lineages include the neurons that comprise the lateral reticular, external cuneate, and inferior olivary nuclei of the precerebellar system which function to connect inputs from the spinal cord and cortex to the cerebellum 5. In order to manipulate expression in the LRL of embryos younger than E12.5, we developed an in vitro system in which embryos are placed into culture following electroporation.
This study presents an efficient and effective method for manipulating the gene expression of LRL progenitors at E11.5. Embryos electroporated with green fluorescent protein (GFP) driven from the broadly active CAG promoter reproducibly expressed GFP after 24 hours of culture. A critical aspect of this assay is that gene expression is only altered because of the expression of the exogenous gene and not because of secondary effects that result from the electroporation and culturing techniques. It was determined that the endogenous gene expression patterns remain undisturbed in electroporated and cultured embryos. This assay can be utilized to alter the fate of cells emerging from the LRL of embryos younger than E12.5 through the introduction of plasmids for overexpression or knock down (through RNAi) of different pro-neural transcription factors.

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

This study describes the development of an in vitro electroporation technique that allows for the manipulation of gene expression in the lower rhombic lip of midgestation embryos.

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior