Name: a pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior
Uploaded: 2020-05-21T14:45:00
Description: Watch this Scientific Journal Video about A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior at JoVE.com

We thank Lisa Kretsge for critical feedback and editing to the manuscript. We thank all research assistants in the Cruz-Mart&#237;n lab who were invaluable in helping with perfusions and cell counting of behavior brains. We thank Andrzej Cwetsch for input on the design of the tripolar electrode, and Todd Blute and the Boston University Biology Imaging Core for use of the confocal microscope. This work was supported by a NARSAD Young Investigator Grant (AC-M, #27202), the Brenton R. Lutz Award (ALC), the I. Alden Macchi Award (ALC), the NSF NRT UtB: Neurophotonics National Research Fellowship (ALC, #DGE1633516), and the Boston University Undergraduate Research Opportunities Program (WWY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All experimental protocols were conducted according to the National Institutes of Health (NIH) guidelines for animal research and were approved by the Boston University Institutional Animal Care and Use Committee (IACUC).
1. DNA solution preparation
<ol>
	<li>Purchase a commercial plasmid or subclone a gene of interest into plasmid with desired promoter. Here, a plasmid containing EGFP under the CAG promoter (pCAG-EGFP) was used. 
	NOTE: Determine the desired promoter based off the leve...

<ol>
	<li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+association+and+meta-analysis+of+a+schizophrenia+GWAS+variant+rs10489202+in+East+Asian+populations.">Genetic association and meta-analysis of a schizophrenia GWAS variant rs10489202 in East Asian populations.</a> Translational Psychiatry. 8 (1), 144 (2018).</li><li>Schizophrenia Working Group of the Psychiatric Genomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+insights+from+108+schizophrenia-associated+genetic+loci.">Biological insights from 108 schizophrenia-associated genetic loci.</a> Nature. 511 (7510), 421-427 (2014).</li><li>Howard, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+meta-analysis+of+depression+identifies+102+independent+variants+and+highlights+the+importance+of+the+prefrontal+brain+regions.">Genome-wide meta-analysis of depression identifies 102 independent variants and highlights the importance of the prefrontal brain regions.</a> Nature Neuroscience. 22 (3), 343-352 (2019).</li><li>Grove, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+common+genetic+risk+variants+for+autism+spectrum+disorder.">Identification of common genetic risk variants for autism spectrum disorder.</a> Nature Genetics. 51 (3), 431-444 (2019).</li><li>Comer, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+schizophrenia-associated+gene+C4+leads+to+hypoconnectivity+of+prefrontal+cortex+and+reduced+social+interaction.">Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction.</a> PLoS Biology. 18 (1), 3000604 (2020).</li><li>Chini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+and+Rescuing+Developmental+Miswiring+in+a+Mouse+Model+of+Cognitive+Impairment.">Resolving and Rescuing Developmental Miswiring in a Mouse Model of Cognitive Impairment.</a> Neuron. 105 (1), 60-74 (2020).</li><li>Clifton, N. 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H., Adikaram, P., Pandey, M., Genis, A., Simonds, W. 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Herein, a pipeline is described that combines the manipulation of novel genes of interest in large populations of frontal cortical neurons with behavioral assays in mice. Moreover, this pipeline allows for the longitudinal study of behavior in the same mice both during early postnatal development and in adulthood. This technique bypasses the need to rely on genetic animal models that can be costly in terms of time and expenses. The strength of this protocol is that it can be used to study neurodevelopmental and neuropsyc...

Genome-wide association studies (GWAS) have driven the discovery of novel candidate genes that are associated with brain pathologies1,2,3,4. These studies have been particularly beneficial in understanding devastating neuropsychiatric disorders such as schizophrenia (SCZ), where the investigation of novel genes has served as a launching point for new lines of research and therapeutic intervention5,6. Genes harboring risk for SCZ show biased expression in the prefrontal cortex (PFC) during prenatal and early postnatal development, a region implicated in the pathology of several neuropsychiatric disorders7. Additionally, mouse models of psychiatric disorders exhibit abnormal activity in PFC networks6,8,9. These results suggest that SZC-associated genes might play a role in the developmental wiring of this region. Further investigation using animal models is required to understand the contribution of these candidate genes to the establishment of connections in the PFC and to determine whether these genes have a causative role in the pathogenesis of neuropsychiatric disorders. Genetic manipulation techniques in mice that allow for the study of gene expression changes on specific neuronal circuits during prenatal and early postnatal development are a promising method to understand the molecular mechanisms that link gene expression changes to PFC dysfunction.
Genetic mouse lines offer a method to study the impact of particular genes on brain development and function. However, relying on transgenic mice can be limiting since there are not always commercially available lines to examine the effects of specific genes on developing neural circuits. Moreover, it can be extremely costly and time consuming to develop custom mouse lines. The use of intersectional genetic manipulation strategies that combine transgenic mice with viral approaches has revolutionized the understanding of the brain10,11,12. Despite much progress, viral strategies come with certain limitations depending on the viral vector type, including limits in packaging capacity that can restrict viral expression13 and cell toxicity associated with viral expression14. Furthermore, in most experimental conditions, robust gene expression using adeno-associated virus (AAVs) requires approximately 2 to 4 weeks15, making routine viral strategies unfeasible to manipulate genes during early postnatal development.
In utero electroporation (IUE) is an alternative approach that allows for a rapid and inexpensive gene transfer16,17 that, when coupled with fluorescent labeling and pharmacogenetic or optogenetic approaches, provides a powerful platform to dissect the function of neuronal circuits. Additionally, with the development of CRISPR-Cas9 genome editing genes can be overexpressed or precisely altered through cell-type specific knock-down or knock-out of specific genes or through the modulation of promoters18,19. Gene manipulation approaches using IUE are especially advantageous when the effect of genes on neuronal circuits need to be tested during narrow developmental windows20. IUE is a versatile technique and overexpression can be easily accomplished by inserting a gene into an expression vector under a specific promoter. Additional control of gene expression can be achieved by driving expression using promoters of different strengths or using inducible promoters capable of temporally controlling gene expression21,22. Additionally, IUE allows for the targeting of cells within specific cortical layers, cell types and brain regions, which isn&#8217;t always feasible using other approaches5,17. Recent advances in the IUE configuration based on the use of three electrodes, which generates a more efficient electric-field distribution, have expanded the functional repertoire of this method and allowed scientists to target new cell types and increase the efficiency, accuracy, and number of cells that can be targeted23,24. This technique was recently used to determine the causative role of complement component 4A (C4A), a gene linked to SCZ, in PFC function and early cognition5.
Presented here is an experimental pipeline that combines gene transfer approaches to target large populations of excitatory neurons in the frontal cortex, including the PFC, with behavioral paradigms that not only enables the study of cell and circuit-level changes, but also allows behavior to be monitored throughout early postnatal development and adulthood. First described is a method to bilaterally transfect large populations of layer (L) 2/3 pyramidal neurons in frontal cortical regions. Next, tasks to assay social behavior in juvenile and adult mice are outlined. Cell counts can be obtained upon the completion of behavioral tasks to quantify the extent and location of cell transfection. Furthermore, the number of cells transfected can be correlated with behavioral data to determine if a greater number of transfected cells leads to greater perturbations in behavior.

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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">13mm Silk Black Braided Suture</td>
			<td style="width:121px;">Havel&#39;s</td>
			<td style="width:161px;">SB77D</td>
			<td style="width:167px;">Suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adson Forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11006-12</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">C270 Webcam</td>
			<td style="width:121px;">Logitech</td>
			<td style="width:161px;">N/A</td>
			<td>Record behavior</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Electroporator</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>See Figure 1 and 2 and Bullmann et al, 2015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-500 Spin Column Plasmid DNA Maxi-preps Kit, 20preps</td>
			<td>Bio Basic Inc.</td>
			<td style="width:161px;">BS466</td>
			<td>Pladmid preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fast Green FCF</td>
			<td style="width:121px;">Sigma</td>
			<td>F7252-5G</td>
			<td>Dye for DNA solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Fine scissors- sharp</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">14060-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand Gauze Sponges</td>
			<td>Fisher Scientific</td>
			<td>1376152</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gaymar Heating/Cooling</td>
			<td style="width:121px;">Braintree</td>
			<td style="width:161px;">TP-700</td>
			<td style="width:167px;">Heating Pad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipette puller</td>
			<td>Sutter Instrument,</td>
			<td style="width:161px;">P-97</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass pipettes</td>
			<td>Sutter Instrument,</td>
			<td>BF150-117-10</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Hair Removal Lotion</td>
			<td style="width:121px;">Nair</td>
			<td style="width:161px;">N/A</td>
			<td>Hair removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">13002-10</td>
			<td>IUE</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Open field maze- homemade acrylic arena</td>
			<td style="width:121px;">Custom-built</td>
			<td style="width:161px;">N/A</td>
			<td>50 &#215; 50 &#215; 30 cm length-width-height</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">pCAG-GFP</td>
			<td style="width:121px;">Addgene</td>
			<td>11150</td>
			<td>Mammalian expression vector for expression of GFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Picospritzer III</td>
			<td>Parker Hannifin</td>
			<td style="width:161px;">N/A</td>
			<td>pressure injector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retractor - 2 Pronged Blunt</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">17023-13</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ring forceps</td>
			<td style="width:121px;">F.S.T.</td>
			<td style="width:161px;">11103-09</td>
			<td>IUE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sterilizer, dry bead</td>
			<td style="width:121px;">Sigma</td>
			<td>Z378569</td>
			<td>sterelize surgical tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUTURE, 3/0 PGA, FS-2, VIOLET FOR VET USE ONLY</td>
			<td>Havel&#39;s</td>
			<td>HJ398</td>
			<td>Suture muscle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water bath</td>
			<td style="width:121px;">Cole-Parmer</td>
			<td>EW-12105-84</td>
			<td style="width:167px;">warming sterile saline</td>
		</tr>
	</tbody>
</table>

a pipeline using bilateral in utero electroporation to interrogate genetic influences on rodent behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

Successful development and implementation of a custom-built electroporator and three prong- electrode. 
For IUEs, an inexpensive custom-built electroporator was built based on a previously described design27 (Figure 1A and Figure 2). A three prong electrode was made23,24 using plastic forceps with 2 negative electrodes attached to the tips of the prongs and t...

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A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

Using this method can allow for the discovery of how certain gene expression changes can attribute to pathologies associated with neurological diseases. The advantage of this technique is that it bypasses the need to create or purchase genetic animal lines which can take years to create and validate. This technique allows for the study of genes implicated in neurological disorders.Determining how specific genes contribute to pathology is an important step in developing novel therapies. The advantage of this protocol is learning to manipulate the embryos so that the experimenter accomplishes the gene transfer step without decreasing the survival rates. Visual demonstration of this method is important because the embryos are very fragile so it is helpful to observe how an experienced experimenter manipulates them during the procedure.Bring pregnant dams to the surgery area at least 30 minutes prior to the surgery. Sterilize the entire surgery site using sterilized germicidal wipes and then 70%ethanol. Then sterilize autoclave tools in a glass bead sterilizer.Transfer sterile PBS to 50 milliliter conical tubes and place the tubes in a water bath heated to 38 to 40 degrees Celsius. Check the sterile saline temperature with a thermometer. Turn on the water heating circulation pump so that it is warmed to 37 degrees Celsius.Then turn on the pressure injector and electroporator and ensure proper function. Briefly spin the plasma DNA solution on a tabletop centrifuge and place it on ice. Pull glass pipettes on a pipette puller so that the tip of the pulled glass pipette is about 50 micrometers in diameter.Then fill the pipette with 20 to 40 microliters of DNA solution. Set up all necessary items for surgery including hair removing lotion, iodine, 70%ethanol, cotton swabs, eye drops, sutures, and gauze. Prepare a surgery sheet and fill out the necessary information.Weigh the mouse prior to surgery and record the weight. After anesthetizing the mouse, administer preoperative analgesics and remove the fur from the abdomen using hair removal cream or a razor. Sterilize the abdomen by swabbing it with povidone iodine and 70%ethanol three times.Create a sterile field around the abdomen with sterile gauze and draping. Make a midline incision in the abdominal skin while lifting the skin up with forceps to avoid cutting through the muscle. Then lift the muscle up and cut through it taking care to avoid cutting vital organs.Carefully pull the uterine horns out of the dam using ringed forceps and place them gently onto the sterile field making sure that they are supported with padding and aren't tugging too far away from the dam. Keep the uterine horn moistened throughout the rest of the surgery with the prewarmed sterile PBS. Position an embryo with either forceps or fingers, then carefully insert the pulled glass pipette into the lateral vertical and inject two to three microliters into each ventricle by inserting the pipette into one and then the other ventricle.The ventricle has been successfully targeted if a crescent shape is present after injection. To transfect cells bilaterally in the frontal cortex, position the two negative electrodes on the sides of the embryo's head just lateral and slightly caudal to the lateral ventricles and position the positive electrode between the eyes just in front of the developing snout. Ensure the embryo is generously moistened and apply four square pulses.Inject and electroporate all embryos, going one by one so that each embryo is electroporated immediately after the DNA solution injection. Once all embryos have been electroporated, carefully insert the uterine horns back into the abdominal cavity. Coat the abdominal cavity in sterile PBS to aid uterine horn placement.Fill the abdominal cavity with sterile PBS so that no air pockets remain after suturing is complete. Suture the muscle with absorbable sutures and the skin with silk non-absorbable sutures. Allow the dam to fully recover in a heated chamber for at least one hour.Check on the dams regularly in the next 48 hours. As the dam recovers from the anesthesia and regains consciousness, it will start moving and whisking. This method was used to transfect approximately 5, 000 layer 2/3 pyramidal neurons with the pCAG-GFP plasmid.Most of these cells were localized to the frontal cortical regions. A representative example shows the rostral caudal distribution of transfected neurons in an adult control mouse, confirming the ability of bilateral IUEs to target and genetically label large populations of pyramidal neurons in the frontal cortex. The first part of the maternal interaction task tests the ability of control P18 transfected mice to find nest bedding.Control mice spent more time exploring their nest bedding than exploring fresh bedding, suggesting that they have intact sensory motor abilities and exploratory behavior. The second part of the task takes advantage of the tendency of mice to be motivated to interact with their mother. In this task, pups spent most of their time near their mother while spending significantly less time exploring the empty cup or nest bedding.Adult control mice spent approximately 35%of the time exploring a novel object. When presented with a novel and a familiar object, adult mice spent more time exploring the novel object. In the sociability task, control adult mice spent similar amounts of time exploring a novel mouse and an empty cup.This behavior was automatically tracked with DeepLabCut software by labeling various points on a mouse, including the limbs, centroid, head, and ears. The software was also used to determine when mice were rearing by examining the length of the mouse's body since this distance becomes shorter when the mouse rears. Following this procedure, many cellular molecular questions can be answered by examining the tissue and assaying different measures such as spine density or expression levels of various proteins.

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