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 &mu;L of DNA and mix with 5 &mu;l 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 regar...

<ol>
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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 border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Cryostat</td>
 <td>Leica</td>
 <td>CM-1850</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biologie tip Dumoxel treated DUMONT forceps</td>
 <td>Fine Scientific Tools</td>
 <td>11252-30</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>20 mm MORIA perforated spoon</td>
 <td>Fine Scientific Tools</td>
 <td>10370-17</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ECM 830 Square Wave Electroporation Generator</td>
 <td>BTX (VWR)</td>
 <td>47745-928</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Harvard Apparatus 7 mm Tweezertrodes* Electrodes </td>
 <td>BTX (Fisher)</td>
 <td>BTX450165 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fisher Isotemp CO2 Incubator</td>
 <td>Fisher</td>
 <td>1325525 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NAPCO CO2 Gas Regulator</td>
 <td>Fisher</td>
 <td>15497020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>12 Well Tissue Culture Plates</td>
 <td>BD Falcon (Fisher)</td>
 <td>877229</td>
 <td>&nbsp;</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>&nbsp;</td>
 </tr>
 <tr>
 <td>HyClone* Donor Equine Serum</td>
 <td>Thermo Scientific (Fisher)</td>
 <td>SH3007402 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum, Qualified, Heat Inactivated</td>
 <td>Invitrogen</td>
 <td>16140-063</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>cellgro* 10,000 IU Penicillin, 10,000&mu;g/mL Streptomycin </td>
 <td>Mediatech (Fisher)</td>
 <td>MT-30-002-CI </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HyClone* L-Glutamine L-Glutamine; 200mM in 0.85% NaCl </td>
 <td>Thermo Scientific (Fisher)</td>
 <td>SH3003401 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fast-Green</td>
 <td>Fisher</td>
 <td>AC41053-0250 </td>
 <td>0.01%</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

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

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.

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 ...

in-vitro-electroporation-lower-rhombic-lip-midgestation-mouse

Research

JoVE Journal

Neuroscience

10.3K visualizações.  University of Illinois at Springfield. Este estudo descreve o desenvolvimento de um In vitro Técnica de electroporação que permite a manipulação da expressão do gene no lábio inferior rômbico de embriões midgestation.