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&#181;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. 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.

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 ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

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 ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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.

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

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...