Name: electroporation of sliced human cortical organoids for studies of gene function
Uploaded: 2024-11-29T15:00:00.000+00:00
Duration: 7 min 13 s
Description: Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

We are grateful to the facilities of the CRTD and Dresden Concept partners for the outstanding support provided, notably K. Neumann and her team at the Stem Cell Engineering Facility, H. Hartmann and her team at the Light Microscopy Facility, A. Gompf and her team at the Flow Cytometry Facility and Hartmut Wolf of the MPI-CBG workshop for the construction of the electroporation chambers. We thank Joshua Schmidt for his feedback on the manuscript. MA acknowledges funding from the Center for Regenerative Therapies TU Dresden, the DFG (Emmy Noether, AL 2231/1-1), and the Schram Foundation.

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

The authors declare that they have no conflicts of interest.

Human cerebral and cortical organoids have become key models to investigate human neocortex development7,16,17. For modeling of human disorders, isogenic hiPSC lines and organoids have become the gold standard68. However, since the generation of novel iPSC lines is time-consuming and costly, the acute manipulation of cortical organoid models for studies of gene function has been widely applied.
Electroporation of cerebral and cortical organoids14,22,26,46,47,48,49,50,51,55 is easy to implement, and many labs that have traditionally used animal models for functional studies are equipped with the required tools. For injection in vivo, the lateral ventricles are typically targeted34,36,37,39,40,41,42. These are modeled in cortical organoids by ventricle-like structures featuring a central lumen that can likewise be injected. Electroporation of cortical organoids primarily targets the end feet of cells lining the ventricles, typically apical radial glia69. The targeting efficiency varies between experiments, but typically, a few 100 to 1,000 GFP-positive cells can be obtained per organoid after 3-7 days of electroporation. The duration of the cell cycle in human neural progenitor cells is roughly twice as long compared to the mouse, where apical radial glia have a cell cycle length of 14 h and basal progenitor cells of 23 h70. Therefore, after 3 days, mainly apical radial glia and intermediate progenitor cells are GFP-positive, whereas GFP-positive cells are distributed across all zones after 7 days. GFP-signal can still be observed several months after electroporation, with GFP-positive cells mainly representing neurons that are scattered throughout the organoid, allowing us to analyze the morphology of individual cells. Targeting of neural progenitor cells lining the ventricle is feasible during the stages where ventricle-like regions are identifiable under the brightfield microscope. In the current protocol15, this is possible from week 2 to week 12 of cortical organoid development, after which point the ventricles shrink and are no longer visible. For an experienced experimenter, it is feasible to inject 50-80 organoids in one session.
While the progeny of apical radial glia inherit gene manipulation reagents during cell division, the direct targeting of mature cells, such as neurons and astrocytes, is not feasible with the standard electroporation approach. Instead, viral targeting methods have been applied to postmitotic cells in the nervous system33,71. In particular, recombinant adeno-associated viruses (AAVs) are widely used for gene transfer in the nervous system as they are easy to produce and transduce terminally differentiated cells with high efficiency71. Moreover, AAVs can be targeted to specific neuronal sub-populations. Different viral targeting approaches, including the use of adeno, Sendai, and lentiviruses, have already been applied in cerebral and cortical organoids15,25,29,30,32,47,72,73,74.
One limitation in cerebral organoid protocols is the development of a necrotic core due to limited oxygen and nutrient supply. Several strategies have been implemented to improve cell viability in the organoid center, including culture of organoids in spinning bioreactors14,54, co-culture of endothelial cells or bioengineering approaches to model vascularization75,76, organoid transplantation into rodent models18,77 and slicing of organoids15,47. Here, we have used the sliced human cortical organoid protocol, which was shown to display reduced hypoxia in the organoid core and support the expansion of ventricle-like structures and the cortical plate-like region15. In addition, the slicing of cerebral organoids is also beneficial for live imaging of basal radial glia, which is located in the subventricular zone, allowing the generation of cell fate decision maps72. Likewise, live imaging was used to study sliced cerebral organoids cultured at the air-liquid interface, which supports neuronal survival and axonal outgrowth47. We found that the slicing of human cortical organoids also makes these organoid models particularly amenable to electroporation, as ventricle-like structures can easily be identified after slicing. Electroporation of cortical organoids enables the introduction of DNA plasmids, classically used for gene overexpression and, more recently, for delivery of entire plasmid libraries55, and of CRISPR/Cas9 gene editing tools for inactivation of gene function26.
The CRISPR/Cas9 technology has revolutionized the investigation of gene function56,57,58,63,66. In this protocol, we have outlined the application of gRNA/Cas9 ribonucleoprotein complexes36 for acute gene inactivation in human cortical organoids. Initial validation of gRNA efficiency was performed in vitro, first in a test tube and second in iPSCs. We find that these straightforward initial tests aid the selection of promising gRNA pairs, particularly in the absence of working antibodies. As more and more tools for CRISPR/Cas9-mediated gene knockout become characterized, the initial validation steps may be omitted in the future. While Next Generation Sequencing of targeted DNA regions isolated from iPSCs is more quantitative and has higher information content, we find that Sanger sequencing can often offer much faster results during the pre-screening of gRNAs. Ultimately, validation of efficient CRISPR/Cas9 editing in cortical organoids and reduction of protein levels in the cells of interest is the most definitive proof of successful CRISPR/Cas9-mediated gene ablation.
The electroporation of sliced human cortical organoids enables acute genetic manipulation for gain- and loss-of-function studies, visualization of cell morphology and neuronal axons, delivery of plasmid libraries for screening approaches and massive parallel reporter assays, labeling of cells for live imaging and delivery of epigenome editing tools. As such, the electroporation of sliced human cortical organoids is a powerful method that can reveal mechanistic insights into human cortical development, neurodevelopmental disorders, and cortical evolution.

The neocortex refers to the outer covering of the cerebral hemispheres and is a structure that is unique to mammals. The neocortex represents the seat of higher cognitive functions1,2,3,4,5. During development, neural stem and progenitor cells give rise to neurons in a process termed neurogenesis. Functional studies investigating human neocortex development provide the basis for elucidating the mechanisms underlying human neural stem cell regulation, neural pathologies, and human brain evolution2,6,7.
Historically, studies of human brain development relied on descriptive histological approaches using post-mortem tissue, with more recent free-floating tissue culture systems enabling functional investigations with human fetal tissue8,9. Additionally, human fetal brain tissue was shown to have the capacity to self-organize into long-term expanding organoids10. Fetal tissue research has provided important insights into human development11,12, yet restricted tissue availability and ethical considerations limit its widespread application for mechanistic studies of human brain development. In the past decade, protocols have been developed that allow the generation of three-dimensional neural organoids from human pluripotent stem cells (hPSC), including human induced PSC (hiPSC)13,14.
Cerebral organoids display important features of the developing brain, such as the formation of ventricle-like structures, apicobasal polarity, cortical cytoarchitecture, interkinetic nuclear migration during radial glia division, and neuronal migration. Importantly, these new human organoid models recapitulate characteristics of the human developing brain that are not modeled well in the mouse, including evolutionarily relevant key neural progenitor types, in particular basal radial glia (or outer radial glia)7,14,15. Several limitations of early cerebral organoid protocols - such as issues with organoid heterogeneity, limited nutrient supply to the inner core, and varied regional identity-have been addressed in recent protocol advancements and further improvements can be expected in the coming years15,16,17,18,19. Human cerebral and cortical organoids have quickly become key models to study human cortical development20, neurological disorders21,22,23,24 and brain evolution25,26,27,28,29, and to perform large-scale screening approaches30,31,32.
For acute genetic manipulation, two methods have primarily been used in animal models of neocortex development: viral delivery by infection of target cells33 and in utero or in vitro electroporation34,35. Injection of DNA - and, more recently, CRISPR/Cas9 ribonucleoprotein (RNP) complexes36- into the lateral ventricles, followed by electroporation, provides the advantage that specific regions of the brain can be targeted based on the orientation of the electroporation electrodes. Electroporation involves brief electric pulses that temporarily increase cell membrane permeability, allowing the introduction of DNA and other charged molecules into cells. In utero electroporation was first performed in the mouse37, where it rapidly became a widely applied methodology for developmental neurobiology. The method was subsequently also applied to other species, such as the rat38,39 and the ferret40,41,42, a gyrencephalic species used to study neocortex expansion and cortical folding3,43,44,45.
Electroporation has also become an important method in human brain organoid research46. In cerebral organoids, electroporation has been applied to visualize cell morphology and neuronal axons14,47, to deliver gene knockdown reagents22,48,49, and for investigation of gene function by overexpression50. The method is not restricted to human models but has also been applied for genetic modification of primate cerebral organoids50,51. Moreover, the electroporation of cortical organoids generated in a Spin&#160;&#937; spinning bioreactor has been described52.
In this protocol, we outline the electroporation of sliced human cortical organoids15 for studies of gene function in cortical development. Brain region-specific organoids increase reproducibility and consistency, which are critical for the success of quantitative analysis, for example, in disease modeling. In the sliced human cortical organoid protocol15, potent patterning cues are applied during iPSC differentiation to obtain a homogeneous population of dorsal forebrain progenitors, which is followed by the application of culture media that promote tissue growth with fewer instructive signals53,54. Slicing of cortical organoids has been shown to reduce cell death resulting from reduced availability of nutrients and oxygen in the organoid core15. Moreover, slicing supports the development of ventricle-like structures containing abundant basal radial glia, with sustained neurogenesis leading to the formation of an expanded cortical plate-like region15. The repeated slicing in this protocol also makes these cortical organoids particularly suitable to electroporation, as the ventricle lumens can be easily identified and targeted by injection. Electroporation of sliced cortical organoids has been applied to study gene regulatory regions and cortical evolution26,55.
During the electroporation procedure, the injection mix is delivered to the lumen of ventricle-like structures. Upon application of a pulsed electric field, the mix is taken up by apical radial glia that line the ventricle. As apical radial glia divide and give rise to more committed cell types4, the electroporated agents are passed on to basal progenitor cells and neurons. Electroporated progeny are distributed in the ventricular zone (VZ) and subventricular zone (SVZ) after 3 days and span most of the cortical wall, including the cortical plate (CP)-like region, at 7 days post-electroporation during mid-neurogenic stages26,55.
Here, we describe CRISPR/Cas9-mediated gene disruption56 by electroporation in sliced human cortical organoids26. In addition, the electroporation method can also be applied for gene overexpression, visualization of cell morphology and cellular processes by expression of fluorescent proteins, delivery of plasmid libraries for Massive Parallel Reporter Assays (MPRA), delivery of epigenome editing tools and labeling of cells for live imaging.

<table><tbody><tr><td>15 mL PP Centrifuge Tubes, conical</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>4D-Nucleofector Core Unit</td><td>Lonza</td><td></td><td></td></tr><tr><td>4D-Nucleofector X Unit</td><td>Lonza</td><td></td><td></td></tr><tr><td>5 mL polystyrene round-bottom tube with cell-strainer cap</td><td>Corning</td><td>Falcon 352235</td><td></td></tr><tr><td>6-well plate, nunclon treated</td><td>Thermo Fisher</td><td>140675</td><td>For hiPSC culture</td></tr><tr><td>6-well plate, ultra low attachment</td><td>Corning</td><td>3471</td><td>For organoid culture</td></tr><tr><td>A83-01</td><td>STEMCELL Technologies</td><td>72022</td><td>Forebrain medium 1 (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Air compressor</td><td>Aerotec</td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Chicken IgY (IgG) (H+L)</td><td>Jackson Immuno Research</td><td>703-545-155</td><td>Secondary antibody, RRID: AB_2340375; dilution 1:1000</td></tr><tr><td>Alexa Fluor 555 Donkey Anti-Rabbit IgG (H+L)</td><td>Invitrogen</td><td>A-31572</td><td>Secondary antibody, RRID: AB_162543; dilution 1:1000</td></tr><tr><td>Alt-R CRISPR-Cas9 crRNA, 2 nmol</td><td>Integrated DNA technologies</td><td></td><td>Custom design (order as 2 nmol)</td></tr><tr><td>Alt-R CRISPR-Cas9 tracrRNA</td><td>Integrated DNA technologies</td><td>1072532</td><td>5 nmol</td></tr><tr><td>Alt-R S.p. HiFi Cas9 Nuclease V3</td><td>Integrated DNA technologies</td><td>1081060</td><td>100 &amp;micro;g, from Streptococcus pyogenes</td></tr><tr><td>Amphotericin B (Fungizone)</td><td>Gibco</td><td>15290018</td><td>Forebrain medium 2, 3, and 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Anti-GFP primary antibody (chicken, polyclonal)</td><td>Abcam</td><td>ab13970</td><td>RRID: AB_300798; dilution 1:2000</td></tr><tr><td>Anti-PCGF4 primary antibody (mouse, monoclonal)</td><td>Millipore</td><td>05-637&amp;nbsp;</td><td>RRID: AB_309865; dilution 1:300</td></tr><tr><td>ApoTome fluorescent microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma Aldrich</td><td>1043003</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>B27-supplement (+ vitamin A)</td><td>Gibco</td><td>17504044</td><td>Forebrain medium 3, 4&amp;nbsp;&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Banana to Micrograbber Cable Kit</td><td>Harvard Apparatus BTX</td><td>45-0216</td><td></td></tr><tr><td>Biometra TRIO-Thermocycler (PCR machine)</td><td>Analytik Jena</td><td>846-2-070-723</td><td></td></tr><tr><td>Capillaries, borosilicate glass with filament, OD 1.2 mm; ID 0.69 mm; 10 cm length</td><td>Science Products</td><td>BF120-69-10</td><td>Need to be pulled to specific thickness</td></tr><tr><td>CHIR-99021</td><td>STEMCELL Technologies</td><td>72052</td><td>Forebrain medium 2&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Collagenase Type IV</td><td>Gibco</td><td>17104019</td><td></td></tr><tr><td>CRTDi004-A</td><td>Stem Cell Engineering Facility at CMCB DD</td><td>https://hpscreg.eu/cell-line/CRTDi004-A</td><td>Single-cell adapted hiPSC line from healthy donor</td></tr><tr><td>DAPI</td><td>Roche</td><td>10236276001</td><td>1 mg/mL stock, use 1:1000 diluted for IF</td></tr><tr><td>Dibutyryl-cAMP</td><td>STEMCELL Technologies</td><td>73884</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma Aldrich</td><td>D2650</td><td></td></tr><tr><td>DMEM/F-12, HEPES</td><td>Fisher Scientific</td><td>31330095</td><td>Forebrain medium 1, 2, and 3&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Dorsomorphin</td><td>STEMCELL Technologies</td><td>72102</td><td>Forebrain medium 1&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Dumont #55 Forceps, straigth, 11 cm</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>ECM 830 Square Wave Electroporation System</td><td>Harvard Apparatus BTX</td><td>45-2052</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>32205-1L-M</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma Aldrich</td><td>EDS-500G</td><td></td></tr><tr><td>Fast Green FCF</td><td>Sigma Aldrich</td><td>F7252</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>GE Healthcare&amp;nbsp;</td><td>SH30070.03&amp;nbsp;</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter Instrument Co.</td><td>P-97</td><td>For pulling of microcapillaries</td></tr><tr><td>Geneious Prime</td><td>GraphPad Software LLC d.b.a Geneious</td><td>Software version 2024.0.5</td><td></td></tr><tr><td>&lt;em&gt;gLACZ&lt;/em&gt;</td><td>Kalebic et al., 2016; Platt et al., 2014</td><td></td><td>5&#039;-TGCGAATACGCCCACGCGATCGG; underlined nucleotides = PAM</td></tr><tr><td>GlutaMAX</td><td>Gibco&amp;nbsp;</td><td>35050038</td><td>Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)15</td></tr><tr><td>Glycine</td><td>Sigma Aldrich</td><td>G8898</td><td></td></tr><tr><td>&lt;em&gt;gPCGF4 &lt;/em&gt;KO1</td><td>this paper</td><td></td><td>5&#039;-TGAACTTGGACATCACAAATAGG (corresponds to the KO images in Figure 3I+J)</td></tr><tr><td>&lt;em&gt;gPCGF4 &lt;/em&gt;KO2</td><td>this paper</td><td></td><td>5&#039;-ACAAATAGGACAATACTTGCTGG (corresponds to the KO images in Figure 3I+J)</td></tr><tr><td>HEPES</td><td>made in house</td><td></td><td>1 M stock</td></tr><tr><td>Horse Serum, heat-inactivated</td><td>Gibco</td><td>26050088</td><td></td></tr><tr><td>Human GDNF Recombinant Protein</td><td>Thermo Fisher</td><td>450-10</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Human/Mouse/Rat BDNF Recombinant Protein</td><td>Thermo Fisher</td><td>450-02</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Sigma Aldrich</td><td>258148</td><td>To make TrisHCl</td></tr><tr><td>ImmEdge (wax) Pen</td><td>Vector Laboraories</td><td>H-4000</td><td></td></tr><tr><td>Insulin solution human</td><td>Sigma Aldrich</td><td>&amp;nbsp;I9278</td><td>Forebrain medium 3&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>BP2618-1</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Gibco&amp;nbsp;</td><td>10828-010</td><td>Forebrain medium 1&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Laser Scanning Confocal 980 Microscope</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Low Melting Point Agarose, ultra pure</td><td>Thermo Fisher</td><td>16520100</td><td>for embedding of hCOs during vibratome sectioning</td></tr><tr><td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free</td><td>Corning</td><td>354230</td><td>For organoid culture</td></tr><tr><td>Matrigel hESC-Qualified Matrix, LDEV-free</td><td>Corning</td><td>354277</td><td>For hiPSC culture</td></tr><tr><td>MgCl2, 1 M</td><td>Thermo Fisher</td><td>AM9530G</td><td></td></tr><tr><td>Microinjector PicoPump + Foot Switch</td><td>World Precision Instruments</td><td>SYS-PV820</td><td></td></tr><tr><td>Microloader Pipette Tips 0.5 to 20 &amp;micro;L</td><td>Eppendorf</td><td>5242956003</td><td>For loading of glass capillaries</td></tr><tr><td>Mowiol 4-88</td><td>Sigma Aldrich</td><td>81381</td><td></td></tr><tr><td>mTeSR 1</td><td>STEMCELL Technologies</td><td>85850</td><td>Stem cell medium</td></tr><tr><td>N-2 supplement</td><td>Gibco</td><td>17502048</td><td>Forebrain medium 2, and 3&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S5886</td><td></td></tr><tr><td>NaHCO3</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>NEB Next High Fidelity 2x PCR Mastermix&amp;nbsp;</td><td>New England Biolabs</td><td>M0541S</td><td></td></tr><tr><td>Neubauer counting chamber</td><td>Brand</td><td>718605</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>Forebrain medium 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Non-Essential Amino Acids</td><td>Gibco&amp;nbsp;</td><td>11140050</td><td>Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Nuclease-Free Duplex Buffer</td><td>Integrated DNA technologies</td><td>1072570</td><td></td></tr><tr><td>O. C. T. Compound</td><td>Tissue-Tek</td><td>4583</td><td>Embedding medium for frozen tissue specimen</td></tr><tr><td>P3 Primary Cell 4D-Nucleofector X Kit S</td><td>Lonza</td><td>V4XP-3032</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher Chemical</td><td>P/0840/53</td><td></td></tr><tr><td>pCAG-GFP</td><td>Addgene</td><td>11150</td><td></td></tr><tr><td>Peel-A-Way Embedding Mold TruncatedT12</td><td>Polysciences Inc.</td><td>18986-1</td><td>For embedding of hCOs for vibratome and cryo-sectioning</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)&amp;nbsp;</td><td>Gibco</td><td>15140122</td><td>Forebrain medium 1, 2, 3, and 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt; and for stem cell medium after nucleofection</td></tr><tr><td>Petri Dish 60 mm x 15 mm, No Vent, Sterile</td><td>Corning</td><td>BP50-02</td><td></td></tr><tr><td>Petri Dish Platinum Electrode Chamber, 5 mm gap</td><td>Harvard Apparatus BTX</td><td>45-0504</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>made in house</td><td></td><td></td></tr><tr><td>Proteinase K</td><td>Sigma Aldrich</td><td>P2308</td><td>From Titrachium album; 10 mg/mL stock</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28706</td><td></td></tr><tr><td>RHO/ROCK pathway inhibitor (Y-27632)</td><td>STEMCELL Technologies</td><td>72308</td><td></td></tr><tr><td>SB-431542</td><td>STEMCELL Technologies</td><td>72232</td><td>Forebrain medium 2&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr><tr><td>Sodium Citrate Dihydrate</td><td>Sigma Aldrich</td><td>W302600-1KG-K</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma Aldrich</td><td>S7903</td><td></td></tr><tr><td>SuperFrost Plus adhesion slides</td><td>Fisher Scientific</td><td>10149870</td><td></td></tr><tr><td>SZX10 with a KL 300 LED</td><td>Olympus</td><td>SZX10</td><td>Or alternative stereoscope</td></tr><tr><td>Thermo Shaker</td><td>Grant bio</td><td>PSC24N</td><td></td></tr><tr><td>Triton-X</td><td>Sigma Aldrich</td><td>T9284</td><td>a.k.a. Octoxinol 9</td></tr><tr><td>Trizma base</td><td>Sigma Aldrich</td><td>T1503</td><td>To make TrisHCl</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>TrypLE Express Enzyme (1x)</td><td>Thermo Fisher</td><td>12604021</td><td>Dissociation enzmye to make single-cell suspensions</td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P1379</td><td>a.k.a. Polysorbate 20</td></tr><tr><td>Tyrode&#039;s salt</td><td>Sigma Aldrich</td><td>T2145-10x1l</td><td></td></tr><tr><td>Vibrating Microtome (vibratome)</td><td>Leica</td><td>VT1200 S</td><td></td></tr><tr><td>Wide-Bore 1000 &amp;micro;L Universal Fit Filter Tips</td><td>Corning</td><td>TF-1005-WB-R-S</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Gibco&amp;nbsp;</td><td>21985-023</td><td>Forebrain medium 1, 3, and 4&amp;nbsp; (https://doi.org/10.1016/j.stem.2020.02.002)&lt;sup&gt;15&lt;/sup&gt;</td></tr></tbody></table>

electroporation of sliced human cortical organoids for studies of gene function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

CRISPR/Cas9-mediated gene ablation requires the design of gRNAs. Generally, targeting one of the first exons of a gene of interest with a pair of gRNAs spaced 7-11 bp apart (avoiding multiples of 3 bp) works well (Figure 1A). As a first test, the efficiency of template recognition by gRNAs may be interrogated in vitro using a PCR template26,40,62. Efficient targeting and Cas9-mediated cutting are visible in the reduction of the template DNA and the appearance of cleaved DNA products on an agarose gel (Figure 1B). Further gRNA testing may be performed in the iPSC line that is used for cortical organoid generation. This requires the nucleofection of iPSCs, which can be visualized by successful fluorescent reporter expression (Figure 1C). Efficient targeting of the locus of interest can be verified by Sanger sequencing, resulting in a chromatogram that abruptly ends or becomes superimposed around the targeting site (Figure 1D), resulting from a spectrum of indels from double-strand break repair by non-homologous end joining66. Guide RNA efficiency tests in vitro and in iPSC cells aid the selection of efficient gRNA pairs for gene ablation in cortical organoids.
Prior to electroporation, cortical organoids are sliced, which supports the identification of ventricle lumens for injection of the electroporation mix (Figure 2A-C). Injected organoids are then transferred to the electroporation chamber (Figure 2D). For the electroporation, cortical organoids with well-developed ventricle-like structures should be used (Figure 2E), whereas cortical organoids lacking visible structures should be discarded (Figure 2F). The electroporation mix contains a dye that allows the visualization of injected ventricles during the procedure (Figure 2G). After 1-3 days in culture, successfully targeted ventricles are visible by native GFP signal under a fluorescent microscope (Figure 2H).
For in-depth histological analysis, immunohistochemistry of cryosections can be performed. Ideally, multiple targeted ventricles are present within an electroporated cortical organoid (Figure 3A,B). The most optimal electroporations target ventricle-like structures in the outer region of the organoid facing the outside (Figure 3C), as these are typically the most expanded ventricles. In some cases, electroporation may lead to excessive cell death (Figure 3D,E), for example, when plasmid concentrations are too high or electroporation settings are sub-optimal. Electroporations facing the interior of the organoid (Figure 3F) often involve less expanded regions of the ventricle-like structures compared to the exterior. In our experience, it is helpful to focus quantitative analysis on defined regions of the organoid to reduce variability. Sometimes, electroporation may cause disruption of the apical surface (Figure 3G), for example, when the injection volume is too high, causing ventricles to rupture, or when electrical pulses are too strong, leading to cell delamination. Finally, when two adjacent ventricles are targeted (Figure 3H), it may become difficult to assign the boundaries of cortical zones and the origin of targeted cells.
Immunohistochemistry may be used to verify successful CRISPR/Cas9-mediated knockout at the protein level in cells of interest (Figure 3I), exemplified here by targeting the Polycomb Repressive Complex 1 (PRC1) protein PCGF467. Signal intensity may be analyzed in GFP-positive cells in control versus experimental conditions (Figure 3J). Alternatively, if no suitable antibody is available, targeted cells may be isolated by FACS from cortical organoids60 and further analyzed by Sanger or Next Generation Sequencing36. Moreover, the reduction of protein levels may also be analyzed using Western blotting.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67598/67598fig1v5.jpg" /> 
Figure 1: Guide RNA design and validation for acute CRISPR/Cas9-mediated gene knockout. (A) Schematic illustration of guide RNA (gRNA) design strategy, with two gRNAs targeting an exon of a gene of interest. Black arrows indicate PCR primers for template preparation used for gRNA validation. (B) Validation of gRNA efficiency in vitro. PCR-amplified templates were digested with RNP complexes. Upon successful cleavage, two bands of different sizes are visible after gel electrophoresis (left), whereas the template remains uncut for low-efficiency gRNAs (right). (C) Bright-field image (left), GFP fluorescence (middle), and merged images (right) of human induced pluripotent stem cells (hiPSCs) 3 days after nucleofection with the RNP complex and a GFP plasmid. Scale bars: 350 &#181;m. (D) Validation of gRNA efficiency in hiPSCs by Sanger sequencing of amplified DNA from GFP-positive cells. Upon successful cleavage of the double-stranded DNA, sequencing tracks show superimposed signals starting from the gRNA target site (top, KO1). In the case of unsuccessful targeting, the DNA sequence track remains unchanged (bottom, KO2) compared to the LACZ control. <a href="https://www.jove.com/files/ftp_upload/67598/67598fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67598/67598fig2v5.jpg" /> 
Figure 2: Electroporation of sliced human cortical organoids. (A) Schematic illustration showing the workflow for the electroporation of human cortical organoids (hCOs). First, hCOs are sectioned into 500-&#181;m-thick slices. Two days later, the RNP mix containing a GFP expression plasmid and Fast Green is injected into lumens of ventricle-like structures, preferentially in the outer region of hCOs. For electroporation, hCOs are placed in the electroporation chamber. After several days of culture, a GFP signal can be detected in electroporated areas. (B) Images of vibratome sectioning of hCOs. The organoids are embedded in 3% low-melting point agarose in a medium in a block (left). The block is sectioned into 500-&#181;m-thick slices (middle). The sliced organoids are transferred back into the culture dish with a wide-bore pipet tip (right). (C) Images showing the injection of the mix containing the RNP, pCAG-GFP, and 0.1% Fast Green into ventricle-like structures of the organoids. The organoids are submerged in Tyrode&#39;s solution and the thin glass capillary is inserted in the lumen of the center of ventricle-like structures (left). Forceps are used to stabilize the organoids (left and middle). The ventricles on the outside of the organoids are targeted (middle). Injected organoids are transferred to the electroporation chamber, or later the culture dish, with wide-bore pipet tips (right). (D) Images of the electroporation chamber, which is made up of a glass Petri dish and two metal blades that are glued to the glass. The metal blades serve as electrodes and contain holes to attach the cables to connect to the electroporator. The injected side of the hCO, visible through a green shadow, should face the anode (positive pole, red cable) (right). (E) Representative images of hCOs showing well-developed ventricle-like structures. The lighter ring represents the ventricular zone-like area. The darker circle in the center of the ventricle-like structure represents the lumen and should be targeted for injection. (F) Representative images of hCOs lacking well-developed ventricle-like structures, which we recommend not to include for further experiments. (G) Representative images of hCOs after injection of the electroporation mix, in which targeted areas are visible by Fast Green (orange arrows). (H) Representative image of hCO showing native GFP signal 24 h after electroporation. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67598/67598fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67598/67598fig3v5.jpg" /> 
Figure 3: Immunohistochemical analysis of electroporated sliced human cortical organoids. (A) Schematic representing an electroporated human cortical organoid (hCO) with green fluorescent signal. (B)&#160;DAPI staining (grey) and immunofluorescence for GFP (green) of a cryosectioned hCO with multiple electroporated ventricles, 7 days after electroporation. (C-H) Example images of different electroporation outcomes, stained for GFP (green) and nuclei (DAPI, grey). (C) An optimal electroporation with intact nuclei (see insert 1, D), an undisrupted ventricle-like structure, and electroporated cells in the area facing towards the outside of the organoid, which is usually most expanded. (E) Example of hCO displaying massive cell death in the electroporated area (see also insert 2, D), characterized by small, DAPI-dense and distorted nuclei. (F) A successful electroporation directed towards the inside of the organoid, which is often more difficult to interpret due to cortical plate-like areas merging with adjacent ventricles. (G)&#160;Disrupted ventricle-like structures in the center of the electroporated area (orange arrow). (H) An example of hCO containing multiple smaller ventricles that are partially merged and, therefore, difficult to quantify. (I) DAPI staining (grey) and immunofluorescence for GFP (green) and PCGF4 (magenta) 7 days after electroporation of an RNP complex targeting LACZ (gLACZ KO, left) or PCGF4 (gPCGF4 KO, right) for CRISPR/Cas9-mediated knockout. Circled cells highlight the reduced intensity of PCGF4, indicating successful gene ablation. A uniform signal can be seen in the LACZ control (left). (J) The graph shows the quantified PCGF4 intensity after immunofluorescence in GFP-positive cells for the control (gLACZ KO) and the KO of PCGF4 (gPCGF4 KO) relative to median PCGF4 expression in the gLACZ condition. Each dot represents a cell. Data from 3-4 organoids from two different batches (grey and black) of the same hiPSC line. **** p &#60; 0.0001, Mann-Whitney test. All images were acquired with a laser scanning confocal (B, C) or fluorescent (E-I) microscope. The images represent maximum intensity projections of 10-15 &#181;m thick z-stacks. White dashed lines indicate the apical surface facing the ventricle lumen and the outer surfaces of hCOs. Orange dashed lines indicate the VZ/SVZ border in merged ventricles. Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67598/67598fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody/Dye</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>1:2,000</td>
		</tr>
		<tr>
			<td>Mouse anti-PCGF4</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Chicken</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Donkey Anti-Rabbit</td>
			<td>1:1,000</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>1 mg/mL stock; 1:1,000</td>
		</tr>
	</tbody>
</table>
Table 1: List of antibodies and dyes and their dilution used for Immunofluorescence staining.

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

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.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

electroporation-sliced-human-cortical-organoids-for-studies-gene

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

961 Views.  TUD Dresden University of Technology. 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. 

Video: Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

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 ...

Neuroscience

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biology

Genetic Modification of Primate Cerebral Organoids

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

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

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

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

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.

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...

Single Cell Electroporation in Organotypic Brain Slice Cultures: An In Vitro Technique to Deliver Plasmids Inside Targeted Neurons in Brain Hippocampal Slices

Source: Keener, D. G. et al. <a href="https://www.jove.com/video/61662" target="_blank">Single-Cell Electroporation Across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons.</a> J. Vis. Exp. (2020)
This video demonstrates the protocol for single-cell electroporation of genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice cultures. The technique is useful to examine specific molecular and physiological functions of neurons, including cell autonomous mechanisms and transsynaptic protein interactions.

Source: Keener, D. G. et al. Single-Cell Electroporation Across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific ...

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Summary

Explore More Videos

Chapters in this video

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

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

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

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Single Cell Electroporation in Organotypic Brain Slice Cultures: An In Vitro Technique to Deliver Plasmids Inside Targeted Neurons in Brain Hippocampal Slices