We are grateful to the Services and Facilities of the Max Planck Institute of Molecular Cell Biology and Genetics for the outstanding support provided, notably the entire team of the Biomedical services (BMS) for the excellent husbandry of ferrets and J. Peychl and his team of the Light Microscopy Facility. We are particularly grateful to Katrin Reppe and Anna Pfeffer from the BMS for exceptional veterinary support and Lei Xing from the Huttner group for assisting with ferret surgeries.

All experimental procedures were conducted in agreement with the German Animal Welfare Legislation after approval by the Landesdirektion Sachsen (licenses TVV 2/2015 and TVV 21/2017).
1. Preparation for in utero electroporation
<ol>
	<li>Prepare the DNA mixture. In this protocol a final concentration of 1 &#181;g/&#181;L of pGFP is used. Dissolve DNA in PBS and supplement with 0.1% Fast Green to facilitate visualization. Once prepared, mix the DNA mixture by pipetting up and down several times or by finger tapping. Store at room temperature until use. 
	NOTE: For coelectroporations, the DNA mixture is prepared to contain a final concentration of 1 &#181;g/&#181;L of plasmid encoding a gene of interest along with 0.5 &#181;g/&#181;L of pGFP diluted in PBS.</li>
	<li>Prepare the surgery table with all the required tools and instruments.</li>
	<li>Pull glass capillaries using the micropipette puller. Adjust the diameter of the capillary tip by cutting off the distal part of the capillary using forceps as previously described41.</li>
</ol>
2. Preparation of ferrets for surgery
<ol>
	<li>Keep the pregnant jills with embryos at E33 fasting for at least 3 h before the surgery to reduce the risk of vomiting.</li>
	<li>Prior to the surgery sterilize all the tools by autoclaving. Perform the surgery in a specially assigned room in aseptic conditions to eliminate potential sources of contamination.</li>
	<li>Place the pregnant jill in the narcosis box with 4% isoflurane.</li>
	<li>When anesthetized, place the ferret on the operation table with a heat pad and attach the narcosis mask with a constant 2&#8211;3% isoflurane flow to the nose. To ensure the appropriate level of anesthesia, check for the lack of the following reflexes:
	<ol>
		<li>The palpebral reflex by touching the periocular skin</li>
		<li>The flexor reflex by pinching the skin between the 2nd and 3rd, or 3rd and 4th toe of both hind limbs</li>
		<li>If the palpebral reflex is absent and flexor reflex is clearly reduced, check for the pain reflex by pinching one toe of each hind limb. 
		NOTE: Ensure to have a stethoscope at hand to monitor heart rate and rhythm if required.</li>
	</ol>
	</li>
	<li>Inject the ferret subcutaneously with analgesic (0.1 mL of metamizol, 50 mg/kg of body weight), antibiotic (0.1 mL/kg of body weight of 20 mg/kg of amoxicillin + clavulanic acid), and glucose (10 mL of 5% glucose solution).</li>
	<li>Place a drop of eye ointment solution on the eyes to prevent eye dehydration during the surgical procedure.</li>
	<li>Shave the ferret belly using a shaver.</li>
	<li>Clean the skin with water and soap, disinfect the belly with 70% ethanol scrub, disinfect 2x with iodine, and let it dry.</li>
</ol>
3. In utero electroporation of ferrets
<ol>
	<li>Ensure that the surgical area is sterile: place sterile surgical tools on sterile tissues, and change coat and gloves.</li>
	<li>Place a sterile surgical drape on the animal.</li>
	<li>Using a scalpel, surgically open the belly at the linea alba with a ~5 cm long cut.</li>
	<li>Cut the muscle layer using scissors.</li>
	<li>Place gauze swabs around the incision site and wet them with PBS. The gauze swabs will absorb the additional PBS that will be added during the surgery. 
	NOTE: Maintain heat and PBS support throughout the surgery.</li>
	<li>Expose the ferret uterus and place it on the gauze swabs.</li>
	<li>Load a glass capillary with the injection solution using a pipette with a long loading tip. Ensure that the loading volume is approximately between 5&#8211;20 &#181;L depending on the number of embryos and number of different experimental conditions. The average injected volume per embryo is 3&#8211;5 &#181;L. Attach the loaded glass capillary to a holder and connect the other side of the holder to a tube and a mouthpiece.</li>
	<li>Inspect the first embryo and find the head.</li>
	<li>Place the fiber optic light source next to the embryo&#39;s head to facilitate visualization. 
	NOTE: Ferret uterine walls are very dark, and it is difficult to see through them unless light is shined directly on them.</li>
	<li>Perform a single intraventricular injection into the ventricle of one of the cerebral hemispheres and keep the other hemisphere intact to serve as an internal control. The ventricle itself is not easily distinct by eye, so its location is estimated based on the location of the pigmented iris, which is visible. The intraventricular injection is done by taking the holder attached to the glass capillary and penetrating the skin, skull, and cerebral tissue with the tip of the glass capillary. 
	NOTE: Make sure to not damage the placenta, which is darker than murine placentas.
	<ol>
		<li>When the tip of the glass capillary is in the ventricle, inject approximately 3&#8211;5 &#181;L of the injection solution by mouth-pipetting using the mouthpiece connected to the glass capillary holder. Because the injected solution contains 0.1% Fast Green, the injected ventricle will now turn dark green, and will be visible as a kidney-shaped structure.</li>
	</ol>
	</li>
	<li>Place the tweezer electrodes on the uterus above the embryo&#39;s head so that the positive pole is placed above the area that will be targeted and the negative pole below the injected area.</li>
	<li>Set the following electroporation conditions on the electroporator: Pulse length = 50 ms; Pulse voltage = 100 V; Pulse interval = 1 s; Number of pulses = 5. Then, press the &#34;Pulse&#34; button on the electroporator.</li>
	<li>Quickly drop several drops of warm 1x PBS on the electroporated embryo.</li>
	<li>Repeat the procedure for all the embryos. Keep the uterus constantly wet with warm PBS. 
	NOTE: If the first embryos facing the vagina are not easily accessible, it is best to not electroporate them.</li>
	<li>When all the embryos are electroporated, place the uterus back into the peritoneal cavity.</li>
	<li>Suture the muscle layer with the peritoneum using a 4-0 suture.</li>
	<li>Suture the skin using the same thickness and spray the wound with aluminum spray.</li>
	<li>Place the animal in a cage and keep it warm with a heat source. Carefully monitor the animal until it wakes up.</li>
</ol>
4. Postoperative care and housing of targeted animals
<ol>
	<li>For 3 days after the surgery ensure that the animals are undergoing the following postoperative care: 20 mg/kg amoxicillin (antibiotic) 1x daily; 25 mg/kg metamizol (analgesic) 3x daily.</li>
	<li>Ensure that the ferrets are housed individually.</li>
	<li>Ensure that the ferrets are examined by a veterinary at least 1x per day.</li>
	<li>Ensure that the ferrets are disturbed as little as possible during delivery.</li>
	<li>After the pups are weaned or sacrificed, prepare the jills for hysterectomy.</li>
</ol>
5. Hysterectomy of ferrets
NOTE: A hysterectomy is performed to reduce the number of sacrificed animals so that the sterilized animals can be donated for adoption as pets. The presurgical procedure for hysterectomy is the same as in step 2.
<ol>
	<li>Shave and sterilize the ferret belly as described in steps 3.1 and 3.2.</li>
	<li>Surgically open the belly at the linea alba. Cut the muscle layer. Lift the muscle layer and shelter the gut with a finger while fully opening the muscle layer.</li>
	<li>Place gauze swabs around the incision site and wet them with sterile PBS. Expose the ferret uterus and the ovaries and place them on the gauze swabs.</li>
	<li>Start the hysterectomy on one side by ligating the arteria ovarica and vena ovarica cranial to the mesovar. Put a clamp on each side of the ligation and perform another ligation cranial to the clamps. Attach the third clamp at the ends of the ligation to save them. Repeat on the other side.</li>
	<li>Cut between the clamps and detach the cornua uteri from the mesentery on both sides.</li>
	<li>Ligate the arteria uterina on both uterine sides caudal to the ostium uteri. Then ligate the vagina and fix the ligature. Attach the clamp at the ends of the ligations to save them. Put two clamps cranial to the ligature. Cut between the clamps and detach the uterus from vagina. Remove the uterus and ovaries and dispose of them. Scrape the residual mucous membrane from the uterus butt.</li>
	<li>Detach all the remaining clamps and shorten the ligature ends. Make sure to control for bleeding after the clamps have been removed.</li>
	<li>Suture the muscle layer and the skin as described in steps 3.16 and 3.17. Follow the postoperative protocol as in steps 3.18 and 4.1&#8211;4.3. Keep the animals in the animal facility for at least 2 weeks with regular veterinary visits. After the animals are fully recovered, they can be donated for adoption.</li>
</ol>

The authors have nothing to disclose.

In utero electroporation in ferret is an important technique, with advantages and disadvantages with respect to other methods. There are critical steps and limitations to this method, as well as potential modifications and future applications to keep in mind.
Since the pioneering work of Victor Borrell and colleagues on genetic manipulation of the postnatal ferret neocortex via electroporation or viral injection35,42,<sup cl...

The neocortex is the outer sheet of the mammalian cerebrum and the seat of higher cognitive functions1,2,3,4,5. In order to achieve an acute genetic manipulation in the mammalian neocortex in vivo during the embryonic development, two different methods have been explored: viral infection6 and in utero electroporation7. Both methods allow efficient targeting of neocortical cells but suffer from some limitations. The major advantage of in utero electroporation compared to viral infection is the ability to achieve spatial specificity within the neocortex, which is achieved by regulating the direction of the electric&#160;field.
Since electroporation was first shown to facilitate the entry of DNA into the cells in vitro8, it has been applied to deliver DNA into various vertebrates in vivo. In developmental neuroscience, in utero electroporation of the mouse neocortex was first reported in 20019,10. This method consists of an injection of the DNA mixture in the lateral ventricle of the embryonic brain and subsequent application of the electric field using tweezer electrodes, which allows spatial precision7,11. In utero electroporation has since been applied to deliver nucleic acids in order to manipulate the expression of endogenous or ectopically added genes in the mouse neocortex. Important progress has been made recently by applying the methodology of CRISPR/Cas9-mediated genome editing via in utero electroporation in the mouse neocortex to perform (1) gene disruption in postmitotic neurons12,13 and neural progenitor cells14, and (2) genome15 and epigenome16 editing.
Very soon after the first report in mouse, in utero electroporation was applied to the embryonic rat neocortex17,18. Non-rodents remained a challenge until the first in utero electroporation of ferrets, a small carnivore, was reported in 201219,20. Since then, in utero electroporation of ferrets has been applied to study the mechanisms of neocortex development by labeling neural progenitors and neurons20,21,22,23, manipulating the expression of endogenous genes, including the use of CRISPR/Cas9 technology24, and by delivering ectopic genes21,22,25, including human-specific genes26. Furthermore, in utero electroporation of ferrets has been used to address features of human neocortex development in pathological conditions27,28.
In the context of neocortex development, the advantages of using ferrets as a model organism compared to mice are due to the fact that ferrets better recapitulate a series of human-like features. At the anatomical level, ferrets exhibit a characteristic pattern of cortical folding, which is also present in human and most other primates, but is completely absent in mice or rats4,29,30,31. At the histological level ferrets have two distinct subventricular germinal zones, referred to as the inner and outer subventricular zones (ISVZ and OSVZ, respectively)32,33, separated by the inner fiber layer23. These features are also shared with primates, including humans, but not with mice34. The ISVZ and OSVZ in ferrets and humans are populated with abundant neural progenitor cells, whereas the subventricular zone (SVZ) of mice contains only sparse neural progenitors21,32,35,36. At a cellular level, ferrets exhibit a high proportion of a subtype of neural progenitors referred to as basal or outer radial glia (bRG or oRG, respectively), which are deemed instrumental for the evolutionary expansion of the mammalian neocortex34,37,38. bRG are hence highly abundant in the fetal human and embryonic ferret neocortex, but they are very rare in the embryonic mouse neocortex35,36. Furthermore, ferret bRG shows morphological heterogeneity similar to that of human bRG, far superior to mouse bRG21. Finally, at a molecular level, developing ferret neocortex shows gene expression patterns highly similar to those of fetal human neocortex, which are presumed to control the development of cortical folding, among other things39.
The cell biological and molecular characteristics of ferret bRG render them&#160;highly proliferative, similar to human bRG. This results in an increased production of neurons and development of an expanded and highly complex neocortex34. These characteristics make ferrets excellent model organisms for studying human-like features of neocortex development that cannot be modelled in mice26,40. To take full advantage of the ferret as a model organism the presented method was developed. It consists of in utero electroporation of E33 ferret embryos with a plasmid expressing GFP (pGFP) under the control of a ubiquitous promoter, CAG. The electroporated embryos can then be analyzed embryonically or postnatally. In order to reduce the number of sacrificed animals, female ferrets (jills) are sterilized by hysterectomy and donated for adoption as pets. If the targeted embryos are harvested at embryonic stages, a second surgery is performed and the embryos are removed by a caesarian section, whereas the jills are hysterectomized. If the targeted embryos are analyzed at postnatal stages, the jills are hysterectomized after the pups have been weaned or sacrificed. Hence, a protocol for the hysterectomy of jills is also presented.

<table><tbody><tr><td>1ml syringe</td><td>BD</td><td>309628</td><td>Electroporation</td></tr><tr><td>4-0 Vicryl suture</td><td>Ethicon</td><td>V392ZG</td><td>Surgery</td></tr><tr><td>Aluminium spray</td><td>cp-pharma</td><td>98017</td><td>Surgery</td></tr><tr><td>Amoxicilin+clavulanic acid (Synulox RTU)</td><td>WDT</td><td>6301</td><td>Surgery</td></tr><tr><td>Cappilary holder</td><td>WPI</td><td>MPH6S12</td><td>Electroporation</td></tr><tr><td>Dexpanthenol Ointment solution</td><td>Bayer</td><td>6029009.00.00</td><td>Surgery</td></tr><tr><td>Drape sheet 45x75cm</td><td>Hartmann</td><td>2513052</td><td>Surgery</td></tr><tr><td>Electrode Tweezer, platinum plated 5mm</td><td>BTX</td><td>45-0489</td><td>Electroporation</td></tr><tr><td>Electroporator</td><td>BTX</td><td>ECM830</td><td>Electroporation</td></tr><tr><td>Fast Green</td><td>Sigma</td><td>F7258-25G</td><td>Electroporation</td></tr><tr><td>Ferret Mustela putorius furo</td><td>Marshall</td><td>NA</td><td>Experimental organism</td></tr><tr><td>Fiber optic light source</td><td>Olympus</td><td>KL1500LCD</td><td>Electroporation</td></tr><tr><td>Forceps</td><td>Allgaier instrumente</td><td>08-033-130</td><td>Surgery</td></tr><tr><td>Forceps 3C-SA</td><td>Rubis Tech</td><td>3C-SA</td><td>Surgery</td></tr><tr><td>Forceps 55</td><td>Dumostar</td><td>11295-51</td><td>Surgery</td></tr><tr><td>Forceps 5-SA</td><td>Rubis Tech</td><td>5-SA</td><td>Surgery</td></tr><tr><td>Gauze swabs large</td><td>Hartmann</td><td>401723</td><td>Surgery</td></tr><tr><td>Gauze swabs small</td><td>Hartmann</td><td>401721</td><td>Surgery</td></tr><tr><td>GFAP antibody</td><td>Dako</td><td>Z0334</td><td>Antibody</td></tr><tr><td>GFP antibody</td><td>Aves labs</td><td>GFP1020</td><td>Antibody</td></tr><tr><td>Glass cappilaries (Borosilicate glass with filament, OD:1.2mm, ID: 0.69mm, 10cm length)</td><td>Sutter Instrument</td><td>BF120-69-10</td><td>Electroporation</td></tr><tr><td>Glucose</td><td>Bela-pharm</td><td>K4011-02</td><td>Surgery</td></tr><tr><td>Heat pad</td><td>Hans Dinslage</td><td>Sanitas SHK18</td><td>Surgery</td></tr><tr><td>Iodine (Betadine solution 100 mg/ml)</td><td>Meda</td><td>997437</td><td>Surgery</td></tr><tr><td>Isofluran</td><td>CP</td><td>21311</td><td>Surgery</td></tr><tr><td>Loading tips 20&amp;micro;l</td><td>Eppendorf</td><td>#5242 956.003</td><td>Electroporation</td></tr><tr><td>Metamizol</td><td>WDT</td><td>99012</td><td>Surgery</td></tr><tr><td>Metzenbaum dissecting scissors</td><td>Aesculap</td><td>BC600R</td><td>Surgery</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>Model P-97</td><td>Electroporation</td></tr><tr><td>pCAGGS-GFP</td><td>NA</td><td>NA</td><td>From Kalebic et al., eLife, 2018</td></tr><tr><td>PCNA antibody</td><td>Millipore</td><td>CBL407</td><td>Antibody</td></tr><tr><td>pH3 antibody</td><td>Abcam</td><td>ab10543</td><td>Antibody</td></tr><tr><td>Scalpel</td><td>Aesculap</td><td>294200104</td><td>Surgery</td></tr><tr><td>Shaver</td><td>Braun</td><td>EP100</td><td>Surgery</td></tr><tr><td>Sox2 antibody</td><td>R+D Systems</td><td>AF2018</td><td>Antibody</td></tr><tr><td>Surgical clamp 13cm</td><td>WDT</td><td>27080</td><td>Surgery</td></tr><tr><td>Surgical double spoon (Williger)</td><td>WDT</td><td>27232</td><td>Surgery</td></tr><tr><td>Surgical drape</td><td>WDT</td><td>28800</td><td>Surgery</td></tr><tr><td>Surgical scissors small</td><td>FST</td><td>14090-09</td><td>Surgery</td></tr><tr><td>Suturing needle holder</td><td>Aesculap</td><td>BM149R</td><td>Surgery</td></tr><tr><td>Tbr2 antibody</td><td>Abcam</td><td>ab23345</td><td>Antibody</td></tr><tr><td>Transfer pipette 3ml</td><td>Fischer scientific</td><td>13439108</td><td>Surgery</td></tr><tr><td>Water bath</td><td>Julabo</td><td>TW2</td><td>Surgery</td></tr></tbody></table>

in vivo targeting of neural progenitor cells in ferret neocortex by in utero electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

In utero electroporation of ferrets at E33 resulted in targeting of the neural progenitor cells lining the ventricular surface of the embryonic neocortex (Figure 1). These cells are called apical progenitors and are highly proliferative, giving rise to all other cell types during development. Upon asymmetric division, apical progenitors generated another apical progenitor and a more differentiated cell, typically a basal progenitor (BP), which delaminated from the ventricular surface. BPs mi...

Watch this Scientific Journal Video about In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation at JoVE.com

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

in-vivo-targeting-neural-progenitor-cells-ferret-neocortex-utero

Research

JoVE Journal

Neuroscience

5.9K Views.  Max Planck Institute of Molecular Cell Biology and Genetics. Dit protocol stelt ons in staat om een genetische manipulatie in vivo uit te voeren in een belangrijk diermodel dat ons in staat stelt om de expansie en vouwen van een zich ontwikkelende neocortex te bestuderen. Het belangrijkste voordeel van deze methode is in het gebruik van hoge ruimtelijke en temporele specificiteit in het richten van die neurale stamcellen die de belangrijkste cel type voor de ontwikkeling van de hersenen. Trek vóór het begin van de procedure glazen haarvaten aan een m...

Video: In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat 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 rats 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 E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

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

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 ...

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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 ...

Developmental Biology

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 ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

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