Name: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos (Video) | JoVE Journal
Uploaded: 2012-08-03T12:00:00.000+00:00
Duration: 7 min 53 s
Description: Learn in vitro electroporation for gene expression manipulation in mouse embryonic hind brain progenitors. This method enables targeted DNA delivery to the lower rhombic lip at midgestation stages.

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5 minutes

The authors would like to thank Jane Johnson for the Math1, Ngn1, and Ptf1a antibodies and Connie Cepko for the pCAG::GFP plasmid. This work was funded by NIH R15 1R15HD059922-01.

1. Preparations Prior to Electroporation
<ol>
 <li>Amplify the DNA for electroporation by a maxi prep (Prime-It or Qiagen). The concentration of the DNA should be a minimum of 1 mg/mL for efficient uptake.</li>
 <li>Remove 495 μL of DNA and mix with 5 μl of 0.01% Fast Green in 1 X PBS (phosphate buffered saline) in a microcentrifuge tube. </li>
</ol>
2. Embryonic Harvest
<ol>
 <li>Establish timed matings of CD-1 mice (Harlan). Check for the presence of vaginal plugs and regard the date a vaginal plug is observed as embryonic day (E) 0.5. Embryos will be harvested 11 days after visualizing plug (E11.5).</li>
 <li>Place sterilized tools in 70% ethanol. Treat a laminar flow hood with UV light for at least one hour prior to use. Spray hood, dissecting tray, and dissecting scope with 70% ethanol. Pre-heat 1 X PBS to 37 °C.</li>
 <li>On E11.5 euthanize the female according to conditions approved by the Institutional Committee for the Care and Use of Animals (ICCUA). Place dissecting tray in the laminar flow hood. Spray down the abdomen of the female with 70% ethanol. </li>
 <li>Open the peritoneal cavity and pin the walls to the dissecting tray. Pull out the uterine horns so that it rests within the peritoneal cavity.</li>
 <li>Carefully cut open the wall of the uterine horn. Use a 20 mm spoon (Fine Scientific Tools) to gently remove the embryo in the yolk sac away from the placenta. Place embryos into a 100 mm tissue culture dish prefilled with 10 mL of sterile 1 X PBS that was preheated in 2.2.</li>
 <li>Repeat for all embryos present.</li>
</ol>
3. Electroporation of E11.5 Embryos (Figure 1)
<ol>
 <li>Using a 20 mm spoon, transfer the first embryo to a new 100 mm dish prefilled with 10 mL of sterile 1XPBS that was preheated to 37 °C. </li>
 <li>Use 11 cm forceps with a 0.05 x 0.02 mm tip (Fine Scientific Tools) to carefully remove and discard the yolk sac.</li>
 <li>Position embryo so that its dorsal side is facing up so that it resembles the cartoon in Figure 1A. Use 7 mm electrode paddles (Harvard Apparatus) to gently hold the embryo. The electrodes should be positioned at either side of the neural tube at the level of the hindbrain fourth ventricle. The ventricle is visible to the naked eye but using a dissection microscope can facilitate accurate paddle placement. Positioning of the paddles is critical to determining the region that receives the electroporated DNA. If the lower rhombic lip (LRL) of the dorsal embryonic hindbrain is desired, paddles must be place such that they are directly flanking the widest part of the fourth ventricle opening.</li>
 <li>Use a 1 cc syringe to draw up the plasmid DNA mixed with 0.01% Fast Green. 500 μL of the mixture should be sufficient for the electroporation of at least 8-10 embryos (see 3.5).</li>
 <li>Gently puncture the roofplate overlying the fourth ventricle with a 25G 5/8 tuberculin needle attached to the 1 cc syringe and inject the DNA-dye mixture into the ventricle. Successful injections are characterized by the DNA-dye mixture filling the entire ventricular system (Figure 1C). The amount of DNA-dye mixture typically injected is less than 50 μL. Exact amounts are variable between embryos as a portion of the mixture tends to leak out of ventricle into the PBS surrounding the embryo. An alternate means of delivering the DNA-dye mixture would be through accessing the ventricular system by puncturing the velum overlying the midbrain. Again, successful injections are characterized by the DNA-dye mixture filling the entire ventricular system.</li>
 <li>Deliver five square pulses using an electric pulse generator (BTX) and 7 mm electrode paddles. Each pulse is 50 V lasting 5 ms per pulse with 500 ms between each pulse. The tissue closest to the positively charged electrode will then take up the plasmid.</li>
</ol>
4. Culture of Embryos
<ol>
 <li>In a laminar flow hood, fill the outer wells of a 12-well culture dish with 2 mL of DMEM/F12 media supplemented with 10% fetal bovine serum, 5% equine serum, 1% glutamine, 1% penicillin/streptomycin that was preheated to 37 °C. The culture conditions were adapted from de Diego and colleagues.15</li>
 <li>In laminar flow hood pinch embryos at midsection (below the heart) with forceps and remove posterior portion of embryo. Place the anterior portion into one of the filled wells of the 12-well culture dish. </li>
 <li>Repeat for all embryos. Fill only the outer wells of the 12-well plate to avoid contamination of cultures.</li>
 <li>Culture embryos in a 37 °C incubator with 5% CO2. Expression of the electroporated plasmid should be observable within 24 hr.</li>
 <li>Should longer culture times be desired, fill the out wells of a new 12 well plate with 2 mL of the media used in 4.1. With a sterilized spoon transfer the embryos to a well in the new plate. Place back into the 37 °C incubator. Culture for up to 48 hr is possible.</li>
 <li>When the desired culture time is reached, fix embryos for analysis (see below). </li>
</ol>
5. Preparation of Embryos for Analysis
<ol>
 <li>Rinse embryos in 1 X PBS at 4 °C for five min. Repeat.</li>
 <li>Fix embryos for analysis in 2% paraformaldehyde (PFA) in 1XPBS for 2 hours at 4 °C.</li>
 <li>Rinse embryos in 1 X PBS at 4 °C for five min. Repeat.</li>
 <li>Equilibrate embryos in 30% sucrose in 1 X PBS overnight at 4 °C.</li>
 <li>Embed embryos in Optimal Cutting Temperature (O.C.T) compound using a dry ice/ethanol bath. Embryos can be stored at -20 °C.</li>
 <li>Section embryos on a cryostat (Leica) into 30 μm sections and mount on slides (VWR, Superfrost Plus). Store at -20 °C.</li>
</ol>
6. Immunohistochemistry Analysis
<ol>
 <li>Immunohistochemistry was performed as described in 16. Primary antibody dilutions used for this study include rabbit α-GFP (Invitrogen) 1:2500; mouse α-Mash1 (BD Biosciences) 1:100; rabbit α-Ngn1 (Jane Johnson) 1:5000; rabbit α-Ptf1a (Jane Johnson) 1:2500; rabbit -Math1 (Jane Johnson) 1:100. Incubate slide flat, specimen side up on a staining tray at 4 °C overnight.</li>
 <li>Slides were analyzed on a compound microscope (Olympus BX51).</li>
</ol>
7. Representative Results
A schematic in Figure 1A depicts the electroporation experiment. Figure 1B shows a sagittal view of an E11.5 embryo prior to manipulation. The same embryo following injection of the plasmid containing CAG::GFP in 0.01% Fast Green is shown in Figure 1C and a representative unfixed embryo exhibiting unilateral GFP expression in the dorsal hindbrain 24 hr following culture is shown in Figure 1D. The extent of the area of the LRL that is successfully electroporated is variable and appears to be highly dependent on positioning of the electrodes. In our studies it was found that 52 out of 65 (80%) of the electroporated embryos successfully expressed GFP. Tissue was deemed to be successfully electroporated if it was positive for GFP in localized regions over several sections following fixation and immunohistochemical analysis (see below). Embryos that failed to meet this criteria were scored as unsuccessful attempts at electroporation.
Further assessment of electroporation efficiency can be ascertained by performing immunohistochemistry against GFP on transverse sections of electroporated embryos at the level of the fourth ventricle. Figure 2 shows representative serial sections from an embryo displaying unilateral GFP expression. Figure 2A shows an idealized schematic that illustrates that the left side of the embryo was toward the positive electrode. Transverse sections (at levels represented by the middle cartoon of Figure 2A) reveal localized GFP expression exclusively on the left side of the hindbrain tissue (Figures 2C and 2D). The electroporated area of the LRL did not extend the entire anterior-posterior axis of the neural tube as examination of sections 300 μm rostral or greater to that shown in Figure 2C does not express GFP (Figure 2B).
The utility of this assay for manipulation of gene expression is dependent upon the stability of the expression domains for the endogenous proteins. The LRL has been characterized as possessing unique progenitor domains characterized by the differential expression of proneural transcription factors (reviewed in 1). A subset of these factors (Mash1, Math1, Ngn1, and Ptf1a) were chosen for analysis due to their proposed and/or characterized roles in the specification of precerebellar neural subtypes in the LRL, a subject of future studies 16-18. All four proteins have highly characteristic expression domains in the caudal hindbrain at E11.5 16-18. We observed that embryos that were placed cultures failed to increase in size and also failed to initiate production of choroid plexus epithelium and invagination of the LRL and roofplate, morphological events that occur between E11.5 and E12.5 5. Based on these observations it was determined that normal development in these embryos was halted or grossly delayed and the comparable control for the cultured embryos should be uncultured embryos at E11.5.
To ensure that culture and electroporation do not disturb levels of endogenous proteins, we analyzed four different proteins by immunohistochemistry (IHC) in 34 different embryos that were electroporated and then cultured for at least 24 hr. Table I shows the number of embryos analyzed for each marker and the percentage of the embryos analyzed that retained normal protein levels. Figure 3 shows representative IHC data from two of the proteins analyzed, Mash1 (Figures 3A and 3B) and Math1 (Figures 3C and 3D). We observed that the majority of the embryos retained normal levels of expression following electroporation and culture (Figures 3B and 3D) as compared to the control embryos at E11.5 (Figures 3A and 3C). Importantly, the characteristic expression domains of these proteins were not perturbed.
<img src="/files/ftp_upload/3983/3983fig1.jpg" alt="Neural tube development: Diagram and embryo images, 4v ventricles, fluorescence microscopy." data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Electroporation of Embryos at E11.5. (A) Schematic of the electroporation experiment. An E11.5 embryo is isolated and the expression plasmid in 0.01% Fast Green is injected into the fourth ventricle. The embryo is then flanked by electrode paddles and subjected to a 50 V pulse prior to being placed into culture (B) Sagittal view of an E11.5 embryo prior to injection. (C) The same E11.5 embryo following injection of plasmid in 0.01% Fast Green. (D) Unilateral hindbrain expression of GFP observed in E11.5 embryo following 24 hr of culture. mb- midbrain; 4v- fourth ventricle.
<img src="/files/ftp_upload/3983/3983fig2.jpg" alt="Neuroanatomy diagram: brain sections and fluorescence microscopy of 4v region in transverse view." data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Expression of GFP in Electroporated Tissue. (A) The cartoon on left depicts the placement of electrodes around an E11.5 embryo. Middle cartoon depicts uptake and expression of plasmid encoding GFP on the left side of the embryo. Cartoon on right is schematic of an idealized transverse section taken through the embryo at levels denoted by black lines in middle cartoon. (B−D) Immunohistochemistry for GFP on transverse sections through an electroporated E11.5 embryo after 24 hr of culture. Arrows indicate the roofplate (rp) which traps the secondary antibody. Images are taken at 10X magnification. The relative levels of the sections shown are depicted by the horizontal lines through the middle cartoon in (A). mb- midbrain;l 4v- fourth ventricle; rp- roofplate.
<img src="/files/ftp_upload/3983/3983fig3.jpg" alt="Immunofluorescence results; α-Mash1 and α-Math1 expression, cell culture conditions comparison." data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Expression of Endogenous Proteins in the Lower Rhombic Lip. Immunohistochemistry for Mash1 (A and B) or Math1 (C and D) comparing transverse sections of E11.5 embryos that were not cultured (A, C) with embryos that were electroporated with CAG::GFP and cultured for 24 hr (B and D). Images taken at 10X magnification.
<table border="1">
 <tbody>
 <tr>
 <td>Proneural Transcription Factor Analyzed</td>
 <td>Number of Embryos Analyzed</td>
 <td>Percentage Retaining Normal Expression Patterns</td>
 </tr>
 <tr>
 <td>Math1</td>
 <td>15</td>
 <td>86.7%</td>
 </tr>
 <tr>
 <td>Mash1</td>
 <td>12</td>
 <td>83.3%</td>
 </tr>
 <tr>
 <td>Ngn1</td>
 <td>7</td>
 <td>71.4%</td>
 </tr>
 <tr>
 <td>Ptf1a</td>
 <td>6</td>
 <td>100%</td>
 </tr>
 </tbody>
</table>
Table I. Percentage of Electroporated and Cultured Embryos Retaining Normal Proneural Transcription Factor Domains in the LRL.

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<li>Wang, V. Y., Rose, M. F., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Math1+expression+redefines+the+rhombic+lip+derivatives+and+reveals+novel+lineages+within+the+brainstem+and+cerebellum%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Math1 expression redefines the rhombic lip derivatives and reveals novel lineages within the brainstem and cerebellum.</a> Neuron. 48, 31-43 (2005).</li>
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No conflicts of interest declared.

The in vitro electroporation technique presented in this study is a novel methodology that can be efficiently utilized to manipulate gene expression in embryos younger than 12 days of gestation. Placement of the embryos into culture permits expression of the introduced gene and circumvents the lethality observed when electroporated embryos are allowed to remain in vivo. This technique allows for the manipulation of gene expression in embryonic progenitors that were previously inaccessible for e...

<table><tbody><tr><td>Cryostat</td><td>Leica Microsystems</td><td>CM-1850</td><td></td></tr><tr><td>Biologie tip Dumoxel treated DUMONT forceps</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>20 mm MORIA perforated spoon</td><td>Fine Science Tools</td><td>10370-17</td><td></td></tr><tr><td>ECM 830 Square Wave Electroporation Generator</td><td>BTX (VWR)</td><td>47745-928</td><td></td></tr><tr><td>Harvard Apparatus 7 mm Tweezertrodes* Electrodes </td><td>BTX (Fisher)</td><td>BTX450165</td><td></td></tr><tr><td>Fisher Isotemp CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher Scientific</td><td>1325525</td><td></td></tr><tr><td>NAPCO CO&lt;sub&gt;2&lt;/sub&gt; Gas Regulator</td><td>Fisher Scientific</td><td>15497020</td><td></td></tr><tr><td>12 Well Tissue Culture Plates</td><td>BD Falcon (Fisher)</td><td>877229</td><td></td></tr><tr><td>HyClone Liquid Media DMEM/F-12 (1:1); With L-Glutamine and HEPES; 500mL </td><td>Thermo Scientific (Fisher)</td><td>SH3002301</td><td></td></tr><tr><td>HyClone* Donor Equine Serum</td><td>Thermo Scientific (Fisher)</td><td>SH3007402</td><td></td></tr><tr><td>Fetal Bovine Serum, Qualified, Heat Inactivated</td><td>Invitrogen</td><td>16140-063</td><td></td></tr><tr><td>cellgro* 10,000 IU Penicillin, 10,000&amp;mu;g/mL Streptomycin </td><td>Mediatech (Fisher)</td><td>MT-30-002-CI</td><td></td></tr><tr><td>HyClone* L-Glutamine L-Glutamine; 200mM in 0.85% NaCl </td><td>Thermo Scientific (Fisher)</td><td>SH3003401</td><td></td></tr><tr><td>Fast-Green</td><td>Fisher Scientific</td><td>AC41053-0250</td><td>0.01%</td></tr></tbody></table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle (reviewed in 1). The rhombic lip can be subdivided into the upper rhombic lip (URL) which encompasses rhombomere 1 (r1) and generates neurons of the cerebellum and the lower rhombic lip (LRL) which gives rise to diverse neuronal brainstem lineages 2-4. LRL derivatives include the auditory neurons of the cochlear nuclei and those of the precerebellar nuclei that are involved in regulating balance and motor control 5-8. Neurogenesis from the LRL occurs over a large temporal window that encompasses embryonic days (E) 9.5-16.55, 9. Different neuronal lineages emerge from the LRL as postmitotic cells (or are born) during distinct developmental days during this neurogenic window. 
Electroporation of gene expression constructs can be used to manipulate gene expression in LRL progenitors and can potentially change the fate of the neurons produced from this region 10-12. Altering gene expression of LRL progenitors in the mouse via in utero electroporation has been highly successful for manipulating lineages born on embryonic day E12.5 or later 10, 12-14. In utero electroporations prior to E12.5 have been unsuccessful primarily due to the lethality associated with puncturing the fourth ventricle roofplate, a necessary step in delivering exogenous DNA that is electroporated into the LRL. However, many LRL derived lineages arise from the LRL earlier than E12.5 9. These earlier born lineages include the neurons that comprise the lateral reticular, external cuneate, and inferior olivary nuclei of the precerebellar system which function to connect inputs from the spinal cord and cortex to the cerebellum 5. In order to manipulate expression in the LRL of embryos younger than E12.5, we developed an in vitro system in which embryos are placed into culture following electroporation.
This study presents an efficient and effective method for manipulating the gene expression of LRL progenitors at E11.5. Embryos electroporated with green fluorescent protein (GFP) driven from the broadly active CAG promoter reproducibly expressed GFP after 24 hours of culture. A critical aspect of this assay is that gene expression is only altered because of the expression of the exogenous gene and not because of secondary effects that result from the electroporation and culturing techniques. It was determined that the endogenous gene expression patterns remain undisturbed in electroporated and cultured embryos. This assay can be utilized to alter the fate of cells emerging from the LRL of embryos younger than E12.5 through the introduction of plasmids for overexpression or knock down (through RNAi) of different pro-neural transcription factors.

This study describes the development of an in vitro electroporation technique that allows for the manipulation of gene expression in the lower rhombic lip of midgestation embryos.

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

The overall goal of this procedure is to manipulate the gene expression in embryonic hind brain progenitors through the introduction of plasmid DNA by electroporation at earlier gestational time points. This is accomplished by first isolating embryos at 11.5 days of gestation and orienting them dorsal side up in sterile phosphate buffered saline. The second step is to inject about 50 microliters of the expression plasmid in 0.01%fast green.In a successful injection, the entire ventricular system will be filled with the DNA fast green mixture. Next, the embryo is subjected to an electrical pulse. The side of the embryo closest to the positively charged electrode will take up the plasmid DNA.The other side will serve as an internal negative control. The final step is the placement of the embryos into tissue culture for at least 24 hours. Ultimately, the electro rated gene will be expressed after 24 hours of culture and can be assessed by immunohistochemistry.The advantage of this technique over existing methods is that manipulation of embryonic hind brainin progenitors is possible at earlier gestational time points. This method can help answer key questions in the field, such as how different ural genes can influence the fate of embryonic progenitors. Begin this procedure by sterilizing the dissecting tools and lamina flow hood according to the accompanying manuscript.To harvest the embryos place a euthanized female mouse that is pregnant with pops at gestational day E 11.5 on the dissecting tray in the hood. Next, spray the abdomen of the mouse with 70%ethanol. Open the peritoneal cavity and pin the walls to the dissecting tray.Then pull out the uterine horns so that it rests within the peritoneal cavity. Using tweezers, carefully open the wall of the uterine horn. Use a 20 millimeter spoon to gently remove the embryos in the yolk sac from the placenta.After that, place the embryos in a 100 millimeter tissue culture dish. Prefilled with 10 milliliters of sterile preheated one times PBS. In this step, transfer the first embryo to a new 100 millimeter dish prefilled with 10 milliliters of sterile one times PBS that was preheated to 37 degrees Celsius.Next, carefully remove and discard the yolk sack. Then position the embryo so that its dorsal side is facing up. Gently hold the embryo with the seven millimeter electrode paddles under a dissection microscope.Place the electrodes one on each side of the neural tube at the level of the hind brain fourth ventricle. After that, use a one cc syringe to draw the mixture of plasmid DNA and 0.01%Fast screen in general, 500 microliters of the mixture should be sufficient for the electroporation of at least eight to 10 embryos. Next, gently puncture the roof plate overlying the fourth ventricle with a 25 gauge five eighth tuberculin needle attached to the one CC syringe.Then inject about 50 microliters of the DNA DI mixture into the ventricle. Successful injections are characterized by the DNA DI mixture filling the entire ventricular system. An alternate means of delivering the DNA DI mixture will be through accessing the ventricular system by puncturing the vellum, overlying the midbrain, turn on the pulse generator, then deliver five square pulses using an electrode pulse generator and the seven millimeter electrode paddles the tissue closest to the positively charged electrode.While then take up the plasmid in a lamina flow hood, fill the ulcer wells of a 12 well cultured dish with two milliliters of DME MF 12 media supplemented with 10%fetal bovine serum, 5%equine serum, 1%glutamine, 1%penicillin streptomycin that was preheated to 37 degrees Celsius. Then pinch the embryo at the midsection with a pair of forceps. Remove the posterior portion of the embryo afterward, place the anterior portion into one of the filled wells of the culture dish.Repeat the procedure for all the embryos and fill only the outer wells of the 12 well plate. To avoid contamination of cultures culture, the embryos in a 37 degree Celsius incubator with 5%carbon dioxide for 24 hours to express the electroporated plasmid. If longer culture time is desired, fill the ser wells of a new 12 whale dish with two milliliters of the D-M-E-M-F 12 media.Transfer the embryos with a sterilized spoon to the wells of a new plate. Then place them back in the 37 degrees Celsius incubator and culture them for up to 48 hours to fix the embryos for analysis. Fill a 12 well plate with one times PBS, then transfer the embryos and rinse at four degrees Celsius for five minutes.Next, fix the embryos in 2%paraldehyde in one times PBS for two hours at four degrees Celsius. Then rinse them three times in one times PBS at four degrees Celsius for five minutes. Again, after filling a new 12, well plate with 30%sucrose in one times PBS.Transfer the embryos to those wells and equilibrate overnight at four degrees Celsius. Then embed the embryos in the optimal cutting temperature compound using a dry ice ethanol bath or store them at minus 20 degrees Celsius. At the end section the embryos to 30 micron sections with a cryostat.Mount them on slides and store them at minus 20 degrees Celsius from uni Histochemical. Study later shown here are the representative results of the immunohistochemistry for GFP on the transverse sections of an electroporated E 11.5 embryo after 24 hours of culture. The arrows shown here indicate the roof plate which traps the secondary antibody.The results show that the electroporated area of the lower ROIC lip was restricted and did not extend to the entire anterior posterior axis of the neural tube. Here shows the results from two of the proteins analyzed MASH one and math one. We observed that the majority of the embryos retain normal levels of expression following electroporation and culture as compared to the control embryos at a 11.5 Once mastered, this technique can be completed in one to two hours if performed properly.After watching this video, you should now have a good understanding of how to use the in vitro electroporation assay to manipulate gene expression in the progenitors of the mouse embryonic membrane.

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Research

JoVE Journal

Neuroscience

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

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation.  These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented.  This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In vivo Electroporation of Developing Mouse Retina

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

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

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

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

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

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 describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Learn in vitro electroporation for gene expression manipulation in mouse embryonic hind brain progenitors. This method enables targeted DNA delivery to the lower rhombic lip at midgestation stages.

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

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