Name: In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation (Video) | JoVE Journal
Uploaded: 2020-05-06T14:30:00.000+00:00
Duration: 7 min 3 s
Description: Learn in utero electroporation for genetic manipulation of neural progenitor cells in the developing ferret neocortex. This method enables precise study of brain expansion and folding mechanisms.

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5 minutes

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>

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<li>Fietz, S. A., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+progenitor+expansion%2C+self-renewal+and+neurogenesis-a+polarized+perspective%5BTitle%5D)+AND+%22Current+Opinion+in+Neurobiology%22%5BJournal%5D)">Cortical progenitor expansion, self-renewal and neurogenesis-a polarized perspective.</a> Current Opinion in Neurobiology. 21 (1), 23-35 (2011).</li>
<li>De Juan Romero, C., Bruder, C., Tomasello, U., Sanz-Anquela, J. M., Borrell, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discrete+domains+of+gene+expression+in+germinal+layers+distinguish+the+development+of+gyrencephaly%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">Discrete domains of gene expression in germinal layers distinguish the development of gyrencephaly.</a> EMBO Journal. 34 (14), 1859-1874 (2015).</li>
<li>Kawasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+investigations+of+the+brain+of+higher+mammals+using+gyrencephalic+carnivore+ferrets%5BTitle%5D)+AND+%22Neurosciences+Research%22%5BJournal%5D)">Molecular investigations of the brain of higher mammals using gyrencephalic carnivore ferrets.</a> Neurosciences Research. 86, 59-65 (2014).</li>
<li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+in+utero+electroporation%3A+controlled+spatiotemporal+gene+transfection%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> Journal of Visualized Experiments. (54), e3024(2011).</li>
<li>Borrell, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+gene+delivery+to+the+postnatal+ferret+cerebral+cortex+by+DNA+electroporation%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation.</a> Journal of Neuroscience Methods. 186 (2), 186-195 (2010).</li>
<li>Borrell, V., Kaspar, B. K., Gage, F. H., Callaway, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+evidence+for+radial+migration+of+neurons+by+long-distance+somal+translocation+in+the+developing+ferret+visual+cortex%5BTitle%5D)+AND+%22Cerebral+Cortex%22%5BJournal%5D)">In vivo evidence for radial migration of neurons by long-distance somal translocation in the developing ferret visual cortex.</a> Cerebral Cortex. 16 (11), 1571-1583 (2006).</li>
<li>Johnson, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aspm+knockout+ferret+reveals+an+evolutionary+mechanism+governing+cerebral+cortical+size%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size.</a> Nature. 556 (7701), 370-375 (2018).</li>
<li>Vaid, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+population+of+Hopx-dependent+basal+radial+glial+cells+in+the+developing+mouse+neocortex%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.</a> Development. 145 (20), 169276(2018).</li>
<li>Pilz, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amplification+of+progenitors+in+the+mammalian+telencephalon+includes+a+new+radial+glial+cell+type%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type.</a> Nature Communications. 4, 2125(2013).</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

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...

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 ...

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Research

JoVE Journal

Neuroscience

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

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

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.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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.

Neonatal Pial Surface 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.

Subtype-selective Electroporation of Cortical Interneurons

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.

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

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

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

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

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Learn in utero electroporation for genetic manipulation of neural progenitor cells in the developing ferret neocortex. This method enables precise study of brain expansion and folding mechanisms.

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

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