Name: generation of topically transgenic rats by in utero electroporation and in vivo bioluminescence screening
Uploaded: 2013-09-24T11:45:00
Duration: 8 min 44 s
Description: Watch this Scientific Journal Video about Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening at JoVE.com

The authors thank Tracy Young-Pearse and Atsushi Kamiya for providing plasmids.
This work was funded by NEURON-ERANET DISCover to O.R. and C.K. (BMBF 01EW1003), DFG (Ko 1679/3-1; GRK1033) to C.K., and (DE 792/2-4) to M.A.S.S.

All animal experiments were authorized by the responsible Landesministerium f&#252;r Natur, Umwelt und Verbraucherschutz (LANUV NRW; 87-51.05.2010.A301) in accordance with National and European legislation.
1. In utero Electroporation
This method has been described in detail in JoVE for the rat by Walantus et al.3, as well as Rice et al.4 and is here summarized only briefly. A litter size of 6-8 pups yields a good outcome. There should be at least two non-electroporated embryos in order to increase the overall survival rate (see below).
<ol>
	<li>Prepare DNA-Mixture that contains 1.5 &#956;g/&#956;l of target-Plasmid (shRNA: pENTR-U6; Invitrogen, Eugene, OR / DISC1 overexpression: pCAX21), 0.5 &#956;g/&#956;l Luciferase containing Plasmid (pCAX), 0.5 &#956;g/&#956;l GFP containing plasmid (pCAGGS22) in 1x PBS solution stained light blue with Fast Green Dye.</li>
	<li>Prepare injection needles out of glass capillaries with a Needle Pipette Puller. And sterilize surgical instruments either by autoclaving or incubation with an alcohol-based disinfectant (kodan Tinktur forte).</li>
	<li>Administer a pre-operative dose of buprenorphine (0.05 mg/kg) 15 min before surgery to a pregnant rat 16 days after fertilization (E16). Then anesthetize the animal in an isoflurane chamber.
	<ol>
		<li>Upon anesthesia, place the rat in a supine position on a 37 &#176;C-warmed operation table with breathing mask connected to the anesthesia device, using oxygen setting at 0.4 L/min and isoflurane at 1.8%.</li>
		<li>After shaving the abdomen, disinfect the shaved area three times with kodan Tinktur Forte (an alcohol-based disinfectant).</li>
		<li>Cover the rat with sterile cloths, exposing only the shaved operation field.</li>
	</ol>
	</li>
	<li>Perform in utero electroporation.
	<ol>
		<li>Cut the abdomen with a scissor along the linea alba (~2 cm).</li>
		<li>Expose the uterine horns carefully with a ring forceps.</li>
		<li>Take care to keep the uterus wall wet with warmed sterile PBS during the whole surgery.</li>
		<li>Inject DNA solution with a thin glass needle into one of the lateral ventricle of the embryos.</li>
		<li>Place the 7 mm electrode around the head of the embryo. To hit a cell population of upper cortical layers, perform the IUE at E1623 and position the positive electrode on the hemisphere above the injected ventricle with a slight dorsal/lateral tendency. To target hippocampal cells change the positive electrode placement to the opposite side than the injected ventricle with lateral to slightly dorsal direction (Figure 1).</li>
		<li>Perform electroporation by five 50 msec pulses at 55 V with 950 msec breaks with a square wave pulse electroporator.</li>
		<li>Spare the first embryo at the vaginal end of each uterus horn in order to increase the chances of survival of all embryos.</li>
		<li>Put the uterus horns back into the mother rat.</li>
		<li>Stich the abdominal wall up with an absorbable Vicryl surgical suture material.</li>
		<li>Close the skin with the Vicryl suture material or with suture clips.</li>
		<li>Place the mother rat back into the home cage and keep it warm for 2-3 hr.</li>
		<li>Hold the rats alone in their home cage in the animal facility room and feed ad libitum. They give birth between E22-24.</li>
		<li>Keep the rat pups with their mother for three weeks and separate them afterwards by gender.</li>
	</ol>
	</li>
</ol>
2. Bioluminescence Live Imaging of the Enzymatic Luciferase Reaction
This method is used to analyze the position of the in utero transfected cells. Co-electroporated firefly luciferase cDNA is translated into active luciferase, which upon metabolizing D-luciferin to oxyluciferin, emits a photon (Figure 2). The resulting luminescence can be detected in the brains of positive electroporated young rats in an IVIS Spectrum up to the age of around 35 days postnatally (Figure 4).
In the present study, the luciferase assay and bioluminescence imaging was performed starting at P7. This time point was chosen to allow mother and pups to recover from birth stress. When initially working with the pups at P0, pup survival was severely affected in that the pups were found dead or eaten by the mother.
Initially, rat pups with successful electroporation are identified by a 2D-bioluminescence picture with an exposure time of three minutes. Subsequently, positive pups are used for creating 3D images in order to specify the location of the electroporated area.
<ol>
	<li>Dilute D-luciferin sodium salt in PBS to a concentration of 15 mg/ml and sterilize it by filtration through a sterile syringe filter.</li>
	<li>Weigh the pups.</li>
	<li>Take the pup in one hand with the abdomen on top and stretch the abdomen slightly. Inject 10 &#956;l/g of body weight of luciferin solution intraperitoneal. For older and more agile ones, pre-anaesthetize the pups with isoflurane in the induction chamber before injecting the luciferin.</li>
	<li>Turn on the isoflurane influx of the XGI-8 Gas Anesthesia System within the IVIS Spectrum with 3% isoflurane.</li>
	<li>Put the snout of the animal into the glass nose cones of the Anesthesia System.</li>
	<li>Hold the animal in a prone position until it is in deep anesthesia (2-3 min). Then reduce the isoflurane influx to 1.5%.</li>
	<li>Choose a 2D-bioluminescence measurement to select positive pups from the whole litter. Use the following settings 
	<img alt="Screen 1" src="/files/ftp_upload/50146/50146screen1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/50146/50146screen1large.jpg" target="_blank">Click here to view larger figure</a>.
	<ol>
		<li>Set a checkmark on Photograph with medium binning and F/Stop at 8, the camera takes a photo from above after starting the measurement.</li>
		<li>Set Excitation filter: block.</li>
		<li>Set Emission filter: open.</li>
		<li>Set Binning to medium.</li>
		<li>Set F/Stop at 1.</li>
		<li>Set Stage level A.</li>
		<li>Set luminescence exposure time to 180 sec.</li>
	</ol>
	</li>
	<li>For creation of 3D pictures in order to better quantify the electroporated area from the positive pups, use the following DLIT settings for firefly luciferase. 
	<img alt="Screen 3" src="/files/ftp_upload/50146/50146screen3.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/50146/50146screen3large.jpg" target="_blank">Click here to view larger figure</a>.
	<ol>
		<li>Set a checkmark on Photograph; the camera takes a photo from above after starting the measurement.</li>
		<li>Set a checkmark on Structure, the surface of the animal is scanned by the IVIS prior the bioluminescence measurement.</li>
		<li>Use following Emission filters and exposure time settings until the age of two weeks: 
		Emission filter 1: 590 nm, exposure time 300 sec 
		Emission filter 2: 600 nm, exposure time 240 sec 
		Emission filter 3: 620 nm, exposure time 180 sec 
		Emission filter 4: 640 nm, exposure time 120 sec 
		For rats older than P20 
		Emission filter 1: 600 nm, exposure time 300 sec 
		Emission filter 2: 620 nm, exposure time 300 sec 
		Emission filter 3: 640 nm, exposure time 300 sec 
		<img alt="Screen 3" src="/files/ftp_upload/50146/50146screen3.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/50146/50146screen3large.jpg" target="_blank">Click here to view larger figure</a>. 
		Due to the decrease in signal strength in older animals, the exposure time is enlarged for the three best emission filters.</li>
		<li>Set Stage level B</li>
		<li>Set Binning to medium.</li>
		<li>Set F/Stop at 1</li>
	</ol>
	</li>
	<li>After measurement, mark the rats by an earhole code to differentiate them from each other and to match them to the IVIS Live Imaging pictures</li>
	<li>At the end of the measurement procedure, turn off isoflurane influx and keep the rat on the warmed plate for some minutes before returning it to its cage.</li>
</ol>
3. Analysis of Bioluminescence Images
The generation of 3D images, 3D movies and the quantification of the volume of the signal source is made by the Living Image software pre-installed on the IVIS Spectrum.
<ol>
	<li>Generation of 3D images
	<ol>
		<li>First, reconstruct a surface topography, therefore set threshold between 20-30%. 
		<img alt="Screen 4" src="/files/ftp_upload/50146/50146screen4.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/50146/50146screen4large.jpg" target="_blank">Click here to view larger figure</a>. 
		<img alt="Screen 5" src="/files/ftp_upload/50146/50146screen5.jpg" /> 
		<img alt="Screen 6" src="/files/ftp_upload/50146/50146screen6.jpg" /></li>
		<li>Start DLIT 3D reconstruction with an image threshold of 10% for each wavelength. 
		<img alt="Screen 7" src="/files/ftp_upload/50146/50146screen7.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/50146/50146screen7large.jpg" target="_blank">Click here to view larger figure</a>. 
		<img alt="Screen 8" src="/files/ftp_upload/50146/50146screen8.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/50146/50146screen8large.jpg" target="_blank">Click here to view larger figure</a>. 
		<img alt="Screen 9" src="/files/ftp_upload/50146/50146screen9.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/50146/50146screen9large.jpg" target="_blank">Click here to view larger figure</a>.</li>
		<li>Create 3D movies by using the animate button within the 3D toolbar.</li>
		<li>Choose different orientations of the 3D image, as well as zooming views as key frames and press the &#39;Record&#39; button, recording the changeover from one position/orientation to another. 
		<img alt="Screen 10" src="/files/ftp_upload/50146/50146screen10.jpg" /> 
		<a href="https://www.jove.com/files/ftp_upload/50146/50146screen10large.jpg" target="_blank">Click here to view larger figure</a>.</li>
	</ol>
	</li>
</ol>
4. Behavioral Testing
Behavioral testing was performed in order to determine whether IUE-mediated gene manipulations in the rat might initiate long-term effects that persist into adulthood. In the present particular case, the effect of transient, unilateral full length human DISC1 cortical overexpression after IUE was investigated by testing locomotion in an open field (OF), with and without a low dose of amphetamine, as a specific test for dopamine-related behavior 24. In a similar procedure performed by Niwa et al. in IUE mice using DISC1 knockdown, IUE mice but not controls showed hypersensitivity to amphetamine18.
Rats that were in utero electroporated with a DISC1 overexpressing vector were held under laboratory conditions with 12 hr light from 7 am to 7 pm and were fed ad libitum. At 3 months of age, rats underwent behavioral testing.
For quantifying locomotion as a readout of amphetamine effects, an open-field of a Tru Scan activity system situated in a sound- and light-isolated chamber was used. This system measures the duration time and distance the animal moves, time and distance spent in the margin or center of the open field, as well as rearing behavior25.
<ol>
	<li>On the first day, test after saline injection
	<ol>
		<li>Weigh the animals.</li>
		<li>Inject intraperitoneally 1 &#956;l/g body weight of a saline solution (1x PBS).</li>
		<li>Right after the injection, put the animal into the open-field and start the measurement of the TruScan system. Record for 15 min and subdivide data into 3 x 5 min parts.</li>
		<li>Put the animal back into its home cage.</li>
	</ol>
	</li>
	<li>Second day, test on amphetamine injection
	<ol>
		<li>Weigh the animals.</li>
		<li>Inject intraperitoneally 1 &#956;l/g body weight of a 0.5 mg/ml amphetamine solution.</li>
		<li>Right after the injection put the animal into the open-field and start the measurement of the TruScan system. Record for 15 min and subdivide data into 3 x 5 min parts.</li>
		<li>Return the animal to its home cage.</li>
	</ol>
	</li>
	<li>Analyze locomotion and rearing behavior generated by specific Tru Scan software. Create Graphs with GraphPad (Prism) and calculate statistics by SPSS Statistics software.</li>
</ol>

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The authors of this study do not have financial interest in this study and have not been sponsored by industry for this study. Open access fees were contributed by PerkinElmer Inc. post publication.

Our study demonstrates that IUE is suited to generate adult rats with neurons expressing a transgene in a selective area of the brain and that, as a result of this intervention, these animals exhibit changes in behavior indicating functionality of the performed manipulation. In this study, as an example, rats overexpressing DISC1 unilaterally in a small part of the prefrontal cortex showed hypersensitivity towards amphetamine (Figure 9).
Selecting rats for electroporation success by in vivo bioluminescence imaging was effective in controlling for the inherent variability of IUE cell transfection and was applied to generate groups with a homogenous IUE area of low inter-subject variability for later investigations.
In this study, we were unable to select electroporated pups from the litter by detection of the co-electroporated GFP-induced fluorescence in the newborn animal, even though at the same time and in the same animal a bioluminescence signal of the equally co-electroporated luciferase could be detected after luciferin injection (Figure 6), and the GFP expressing neurons were still present in the brain at an age of six months. We conclude that, in the rat, the luciferase/luciferin reaction is well-suited to differentiate animals with successful electroporated brains (Figure 3).
The quantitative monitoring of IUE success relates to the strength of the bioluminescence signal which is measured by the counts of photons within the same exposure time (Figure 3) and corresponds to the enzymatic activity of co-expressed luciferase. Small bioluminescence signals are detectable by 100-200 counts of photons, and, at a radiance of ~ 1x104 photons/sec/cm2/steradian show 1,000-2,000 GFP-stained cells in the histology in the 6 months old rat brain. The highest signal displayed a radiance of up to ~5x106 photons/sec/cm2/steradian and ~64,000 counts.
In the Sprague Dawley rat strain used, we observed a weakening of the bioluminescence signal longitudinally with increasing age and the signal disappeared beyond the age of P35 (Figure 4). At this point, we do not know whether either the transient, plasmid vector-based expression of luciferase decreases, or if the bioluminescence signal weakens due to increasing brain mass, or both are the causes for the disappearing signal. For the present functional assay in the adult rats, the selection for behavioral studies was merely made based on the location of the signal, but not by bioluminescence signal strength.
Even though 3D quantitative bioluminescence monitoring allowed differentiation between different electroporated areas (Figure 5), its accurateness was limited for cells located in the depth dimension of the brain. Figure 6 shows an example of a hippocampal electroporation where the bioluminescence measurement in the 2D and the 3D picture indicated a good positioning of the electroporation. In the dissected post mortem brain, a GFP-fluorescence signal was detected at about the same position as the bioluminescence signal, indicating correct targeting of the hippocampus. But histology shows that also cells in the cortex dorsal of the hippocampus had been targeted (Figure 7). This indicates that the bioluminescence assay is a useful tool to detect positive, IUE pups and also to have an idea of the electroporated area, but, ultimately, imaging cannot replace post mortem histology to exactly localize positively targeted cells.
Our demonstration indicates promise for the application of the IUE technology to generate subtle targeted manipulations of cortical or hippocampal brain regions to simulate aberrances in cortical migration or other neurodevelopmental defects that may influence the adult animal. While bilateral electroporation26 has the advantage of a likely bigger effect on behavior, there is also more mortality of embryos. Unilateral electroporation was chosen in order to compare the two hemispheres with one as an internal control, as well as for showing that even IUE manipulating in an unilateral, small region is sufficient to change behavior. IUE-induced changes in connectivity or architecture between neurons may thus be induced without evoking a lesion and the required match of the to-be-IUE-manipulated region with the appropriate behavioral test is dependent on the scientific question.
Trouble shooting
Reduced litter size There are several suggestions regarding increasing the survival of the IUE pups. First, the use of very thin glass capillaries during electroporation in order to minimize tissue lesion is recommended. Second, do not electroporate the first embryo at the vaginal end of each uterus horn: death of the first-born embryo increases the chances of an abort of all other embryos. Third, after birth, mother rats often kill part of their progeny due to perinatal stress. In order to reduce additional stress, do not start with the live imaging right after birth, but wait for seven days.
GFP-fluorescence detection of the pups
At one week after birth, no signal of fluorescence by either using live binocular fluorescence microscopic imaging or fluorescence imaging with the IVIS Spectrum (epifluorescence and transfluorescence modes; for GFP excitation/emission: 465/520 nm and 500/540 nm). It is possible that both, the limited transmission of short wavelength excitation and emission light through tissue like the skull and the high autofluorescence background of the skin prevent using fluorescence under the described conditions in the rat. As shown in Figure 6, the luciferase signal in the living animal can also be detected in the dissected brain (without skull) and there, also a fluorescence signal is detectable (Figure 6D).
Differentiation of bioluminescence in closely spaced brain areas
Even in the 3D illustration the location of the bioluminescence area cannot be predicted to 100%. Especially cells on top of or below of the predicted area can also be accidentally targeted and transfected. The exact position has to be controlled by post mortem (fluorescence) histology (see Figure 7).

The development of the in utero electroporation (IUE) method which allows a modulation of gene expression in the developing brain, has been a break-through since it enabled studying neurodevelopment with relative ease.1-7 Changes in expression levels of a target gene in a specific brain region during embryonic and/or perinatal development in rodents were demonstrated to critically influence neuronal proliferation, migration, arborization, and connectivity.8-10
 Schizophrenia is a complex mental illness with acute and chronic symptoms that has been related to neurodevelopmental abnormalities11, 12 and therefore many of the identified candidate genes for schizophrenia are investigated for potential modulating effects on neurodevelopment, like for example for the disrupted-in-schizophrenia-1 (DISC1) gene13-15. 
Brain development is regulated by genetic factors and their interactions with environment which play roles in pre-, peri- and postnatal developmental periods. One major genetic risk factor for various behavioral disorders is the DISC1 16 gene. DISC1 knockdown leads to migration defects in mice 13, 17, and manipulation of DISC1 expression in the developing cortex by IUE has been shown to impact behavior of adult mice18.
 Manipulating brain gene expression by IUE has several advantages19 over the generation of transgenic animal lines. First, gene expression within areas of interest is achieved within weeks to months rather than several generations of breeding transgenic rodent lines. Second, compensatory mechanisms during early development that may shield phenotypes in germline-engineered animals 20 are avoided. Third, through targeting only a specific cell population or specific area of the brain, migration or proliferation differences can be directly compared with the non-mutant or control contralateral side if unilateral electroporations are chosen. On the other hand, IUE does not have the accuracy of promoter-driven cre/lox-induced timing of expression and only a subpopulation of the cells in a certain area is targeted leading to a mosaic kind of gene expression pattern. 
 For many experimental applications in adult rodents, a transient transfection of a limited number of cells in a brain region may be sufficient, or even desired, so that the major advantage of stable, germline-transgenic rodents is negligible. In fact, IUE is useful to investigate whether some abnormally developed cells may affect a whole network of cells or circuitry. Another advantage may be the ability to demonstrate non cell-autonomous effects of a gene due to the mosaic nature of the hit. Furthermore, the generation of transgenic and knockout rats is still in its infancy and the use of IUE in this species for studying aberrant brain development consequences is of high interest.
 So far, a major obstacle of using IUE for investigating the intervention consequences in those animals as adults is the lack of monitoring electroporation success. So far, GFP-co-transfected fluorescent neurons in the live newborn rat pups could not be detected under a suitable binocular fluorescence microscope or with the fluorescence imaging of the IVIS Spectrum. 
 To overcome this obstacle, we co-transfected a luciferase reporter gene and performed bioluminescence live imaging of pups by 3-dimensional (3D) quantitation of the IUE brain area.
 As an example for demonstrating the applicability of this method in a later functional assay testing the neurodevelopmental genetic manipulation, a co-injection of plasmids containing human DISC1, luciferase, and GFP into the lateral ventricle of rat embryos3 followed by electroporation with a tweezer electrode was performed. While fluorescence signals could not be detected in postnatal stages in vivo, a solid bioluminescence signal derived from luciferin metabolism by co-transfected luciferase gene was detected up to five weeks after birth. 3D-measurements of the electroporated brain area allowed quantification whereby pups with insufficient or misplaced electroporation were identified from the outset, thus, enabling the assignment of IUE animals (gene of interest and scrambled control) to experimental groups with matched electroporated brain areas of low variability. The use of adult IUE rats in behavioral paradigms was demonstrated as an example of the usefulness of this protocol.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent name</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline (PBS)</td>
			<td>Invitrogen</td>
			<td>14190-250</td>
			<td>without calcium, without magnesium</td>
		</tr>
		<tr>
			<td>D-luciferin, sodium salt</td>
			<td>SynChem OHG, Germany</td>
			<td>BC218</td>
			<td>CAS number: 103404-75-7 substrate for firefly-luciferase</td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma Aldrich, USA</td>
			<td>F7258-25G</td>
			<td>CAS: 2353-45-9</td>
		</tr>
		<tr>
			<td>D-Amphetamine</td>
			<td>Sigma Aldrich, USA</td>
			<td>A 5880</td>
			<td>CAS: 51-63-8</td>
		</tr>
		<tr>
			<td>kodan Tinktur forte</td>
			<td>Sch&#252;lke &#38; Mayr GmbH, Germany</td>
			<td>104 005</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material / product</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Sutter Instrument</td>
			<td>Novato, California, USA</td>
			<td>borosilicate glass O.D.:1 mm, I.D.: 0.78 mm</td>
		</tr>
		<tr>
			<td>Needle Pipette Puller</td>
			<td>David Kopf Instruments</td>
			<td>Tujunga, California, USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer electrode</td>
			<td>Nepa Gene CO., LTD.</td>
			<td>Shioyaki, Ichikawa, Chiba, Japan</td>
			<td>7 mm in diameter platinum disc electrodes (<a href="http://www.sonidel.com/product_info.php?products_id=129" target="_blank">CUY650P7</a>)</td>
		</tr>
		<tr>
			<td>Surgical Scissors - sharp</td>
			<td>Fine Science Tools</td>
			<td>Heidelberg, Germany</td>
			<td>Straight, 12 cm (14002-12)</td>
		</tr>
		<tr>
			<td>Ring Forceps</td>
			<td>Fine Science Tools</td>
			<td>Heidelberg, Germany</td>
			<td>2.2 mm ID, 3 mm OD (11021-12)</td>
		</tr>
		<tr>
			<td>Square wave pulse electroporator (CUY21SC)</td>
			<td>Nepa Gene CO., LTD.</td>
			<td>Shioyaki, Ichikawa, Chiba, Japan</td>
			<td>(CUY21SC)</td>
		</tr>
		<tr>
			<td>Vicryl surgical suture material</td>
			<td>Ethicon</td>
			<td>Norderstedt, Germany</td>
			<td>3-0; 2 Ph. Eur;</td>
		</tr>
		<tr>
			<td>Wound Clip Applicator</td>
			<td>Fine Science Tools</td>
			<td>Heidelberg, Germany</td>
			<td>Reflex 9 mm (12032-09)</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>VWR</td>
			<td>Darmstadt, Germany</td>
			<td>0.45 &#956;m cellulose acetate</td>
		</tr>
		<tr>
			<td>IVIS Spectrum</td>
			<td>Caliper Life Science / PerkinElmer</td>
			<td>Waltham, MassachusettsUSA</td>
			<td></td>
		</tr>
		<tr>
			<td>XGI-8 Gas Anesthesia System</td>
			<td>PerkinElmer</td>
			<td>Waltham, Massachuset tsUSA</td>
			<td></td>
		</tr>
		<tr>
			<td>Open-field</td>
			<td>Coulbourn Instruments</td>
			<td>Allentown, USA</td>
			<td>(40 x 40 x 39 cm)</td>
		</tr>
		<tr>
			<td>Tru Scan activity system</td>
			<td>Coulbourn Instruments</td>
			<td>Allentown, USA</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="4">Table of recipes</td>
					</tr>
					<tr>
						<td>Number</td>
						<td>Buffer name</td>
						<td>Content</td>
						<td>Comments </td>
					</tr>
					<tr>
						<td>1</td>
						<td>DNA-Mixture for DISC-1 overexpressing</td>
						<td>1.5 &#956;g/&#956;l pCAX humanDISC-1, 
						0.5 &#956;g/&#956;l pCAX-luciferase, 
						10x PBS (10%) Fast Green Dye (0.5%) add H2O</td>
						<td>100 &#956;l of the mixture are enough for ~5 IUE</td>
					</tr>
					<tr>
						<td>2</td>
						<td>DNA-Mixure for control</td>
						<td>0,75 &#956;g/&#956;l control shRNA1, 0,75 &#956;g/&#956;l control shRNA2, 0.5 &#956;g/&#956;l pCAX-luciferase, 0.5 &#956;g/&#956;l pCAGGS-GFP 10x PBS (10%) Fast Green Dye (0.5%) add H2O</td>
						<td>100 &#956;l of the mixure are enough for ~5 IUE</td>
					</tr>
					<tr>
						<td>3</td>
						<td>Fast green Dye</td>
						<td>10 mg/ml Fast Green FCF in ddH2O</td>
						<td></td>
					</tr>
					<tr>
						<td>4</td>
						<td>D-luciferin solution</td>
						<td>15 mg/ml D-luciferin, sodium salt in PBS</td>
						<td></td>
					</tr>
					<tr>
						<td>5</td>
						<td>Amphetamine solution</td>
						<td>0.5 mg/ml D-Amphetamine in PBS</td>
						<td></td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

generation of topically transgenic rats by in utero electroporation and in vivo bioluminescence screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Figure 3 shows live imaging measurements of three rat pups at P7 after the injection of 150 mg luciferin/kg body weight. Differences of signal strength indicating the variability in the efficiency of the IUE are visible. Strong bioluminescence signals were recorded until P36 (Figure 4). In Figure 5, the ability to define cortical (Figures 5A and 5C) and hippocampal (Figures 5B and 5D) electroporation by bioluminescence imaging are depicted. Correlation of the bioluminescence signal (Figure 7A) with its fluorescence signal after skull removal (Figure 7B) and the corresponding GFP-electroporated cells in cryosectioned brain (Figure 8) at P14 are depicted in Figures 6-8. Of note, there was no detection of a fluorescence signal in live rats at any time point.
In vivo bioluminescence imaging enables approximate discrimination of different electroporated brain areas by 2D (Figures 5A and 5B) which is greatly improved with the DLIT program of the IVIS Spectrum software generating 3D images (Figures 5C and 5D). In the shown examples, electroporation of the prefrontal cortex and hippocampal could be distinguished. It should be noted that while aiming to electroporate the hippocampus, some progenitor cells for the cortex lying dorsal of the hippocampus can also be hit (Figure 7).
Rats unilaterally in utero electroporated with pCAX vector into the cortex and subsequently overexpressing full length human DISC1 were investigated for both spontaneous and amphetamine-induced hyperactivity as adults. Rats electroporated with pCAX-DISC1 were hypersensitive to a low dose of amphetamine. These rats moved significantly more after amphetamine treatment than after to saline injection, whereas control animals did not (Figure 10).
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50146/50146fig1.jpg" /> 
Figure 1. Scheme of electrode position for A) cortex electroporation and B) hippocampal electroporation; green = injected DNA-Mix within the ventricle.
<img alt="Figure 2" fo:content-width="4in" src="/files/ftp_upload/50146/50146fig2.jpg" /> 
Figure 2. Luciferase reaction.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/50146/50146fig3.jpg" /> 
Figure 3. Luminescence measurement of P7 rats after injection of 150 mg/kg body weight luciferine; exposure time 180 sec; A) rat with no luminescence signal; B) rat with a weak bioluminescence signal; C) rat with a strong luminescence signal.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/50146/50146fig4.jpg" /> 
Figure 4. Time line of consecutive bioluminescence measurements of the same rat after injection of 150 mg/kg luciferine demonstrating a large time window where IUE success can be detected.
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/50146/50146fig5.jpg" /> 
Figure 5. Illustration of differences between cortical and hippocampal electroporation at P7. A) &#38; B) 2D pictures; C) &#38; D) 3D pictures; A) &#38; C) from cortex; B) &#38; D) from hippocampus. <a href="https://www.jove.com/files/ftp_upload/50146/50146fig5highres.jpg">Click here to view larger figure</a>.
<img alt="Figure 6" fo:content-width="6in" src="/files/ftp_upload/50146/50146fig6.jpg" /> 
Figure 6. Illustration of E16 hippocampal electroporation of a rat pup at P14 A) 2D-picture of bioluminescence B) 3D illustration of the bioluminescence signal C) dissected brain with bioluminescence signal D) brain with GFP epi-fluorescence signal. <a href="https://www.jove.com/files/ftp_upload/50146/50146fig6large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/50146/50146fig7.jpg" /> 
Figure 7. Detail of a fluorescence picture from a 20 &#956;m cryo section of the same P14 rat brain electroporated with GFP containing plasmid and luciferase vector, nuclei staining with DAPI; CA1-3 = Cornu Ammonis 1-3; DG = Dentate Gyrus; FC = Fasciolarum cinereum.
Video 1. 3D animation of a hippocampus electroporated rat at P14.
<img alt="Figure 8" fo:content-width="4in" src="/files/ftp_upload/50146/50146fig8.jpg" /> 
Figure 8. Amphetamine test scheme: first day saline injection before the 15 min trial, 24 hr later amphetamine injection before testing in the open field chamber.
<img alt="Figure 9" fo:content-width="4in" src="/files/ftp_upload/50146/50146fig9.jpg" /> 
Figure 9. Amphetamine test. Bar graph showing moved distance (in cm) of the animal recorded by a TrueScan system over 15 min. White bars = control group; grey bars = DISC1 overexpressing group; control group n = 10; DISC1 overexpression group n = 11; ANOVA: geno*treatment p = 0.043; T-Test for total time saline vs amphetamine n.s.= not significant pcontrol = 0.172, pDISC1 = 0.001.

Watch this Scientific Journal Video about Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening at JoVE.com

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

generation-topically-transgenic-rats-utero-electroporation-vivo

Research

JoVE Journal

Neuroscience

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 ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

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

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.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

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  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.