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 tim...

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 ...

This protocol allows us to perform a genetic manipulation in vivo in a key animal model that allows us to study the expansion and folding of a developing neocortex. The main advantage of this method is in using high spatial and temporal specificity in targeting those neural stem cells that are the key cell type for brain development. Before beginning the procedure, pull glass capillaries on a micro pipette puller and use forceps to cut off the distal part of the capillary to adjust the diameter of the capillary tip.On embryonic day 33, add the appropriate concentration of DNA in PBS, supplemented with 0.1%Fast Green with gentle mixing, and place a 3-hour-fasted, anesthetized, pregnant female ferret on an operation table with a heat pad. Confirm the appropriate level of sedation by touching the periocular skin and pinching the skin between the second and third or third and fourth toes of both hind limbs. Isoflurane can cause adverse effects, including nausea and dizziness.When handling isoflurane in liquid form, use the protective equipment. During the surgery, use appropriate canisters to capture the gas. Inject the animal subcutaneously with analgesic, antibiotic, and glucose, and place ointment on the animal's eyes.Use clippers to shave the abdomen, and clean the exposed skin with a water, soap, and iodine solution. Then, dry the skin with gauze swabs and disinfect with alcohol and iodine scrubs. For in utero electroporation, place a sterile drape over the animal and use a scalpel to make an approximately 5-centimeter skin incision at the linea alba.Use scissors to cut through the muscle layer and place gauze swabs around the incision site. Wet the gauze with PBS and place the uterus onto the swabs. Using a pipette with a long tip, load one of the pulled glass capillaries with 5 microliters of DNA solution per embryo to be injected.Attach the loaded capillary to a holder and connect the other side of the holder to a tube and a mouthpiece. Locate the head of the first embryo and place a fiber optic light source next to the head. Using the pigmented iris as a reference point, penetrate the skin, skull, and cerebral tissue with the tip of the glass capillary and use mouth pipetting to facilitate the delivery of 3-5 microliters of the injection solution into the ventricle of one of the cerebral hemispheres.Because the injected solution contains 0.1%Fast Green, a successful injection will result in a dark green staining of the injected ventricle, which will now be visible as a kidney-shaped structure. After the injection, place the tweezer electrodes on the uterus above the head of the embryo with the positive pole above the area to be targeted and the negative pole below the injected area. Set the pulse length of the electroporate to 50 milliseconds, the pulse voltage to 100 volts, the pulse interval to one second, and the number of pulses to five.When all of the parameters have been set, press Pulse"and quickly drop several drops of warm PBS onto the electroporated embryo. When all of the embryos have been electroporated, return the uterus to the peritoneal cavity and use a 4-0 suture to close the muscle layer with the peritoneum. Close the skin in a similar manner and cover the wound with aluminum spray.Then return the animal to its cage with a heat source and monitoring until full recumbency. Four days after electroporation at embryonic day 37, most of the targeted cells and their progeny are still in the germinal zones and the cells are seldom observed further basally within the cortical plate. The progenitor identity can be examined by immunofluoresence for markers of cycling cells, such as PCNA, whereas a subset of progenitors undergoing mitosis can be shown by markers such as phospho-histone 3.At post-natal day zero, eight days after electroporation, the progeny of the targeted cells spread to all of the histological layers. Using a combination of transcription factor markers, different basal progenitor populations can be revealed. For example, Sox2 is a marker of proliferative progenitor cells, including basal radial glia.And T-box brain protein 2 is a marker of neurogenic basal progenitors, which are mainly intermediate progenitors. By post-natal day 16, a majority of the targeted cells stop dividing and differentiate into neurons and glia, with ferret post-natal day 16 neocortex exhibiting the characteristic folding pattern. The in utero electroporation of ferret embryos is an extremely powerful method to study the function of genes.It has allowed us to study genes that have been implicated in the expansion of the neocortex in evolution, including human-specific genes. And using this powerful method, we have indeed been able to find out key features of how the evolutionary expansion of the human neocortex likely worked.

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Research

JoVE Journal

Neuroscience

5.8K vistas.  Max Planck Institute of Molecular Cell Biology and Genetics. Este protocolo nos permite realizar una manipulación genética in vivo en un modelo animal clave que nos permite estudiar la expansión y plegado de un neocórtex en desarrollo. La principal ventaja de este método es el uso de alta especificidad espacial y temporal en la orientación de las células madre neurales que son el tipo de célula clave para el desarrollo cerebral. Antes de comenzar el procedimiento, tire de los capilares de vidrio en un tirador de micro pipetas y utilice fórceps...