We thank Ekaterina Karelina for help with soundtrack recording for the video, Ivan Molotkov for 3D animation and Dr. Peter Blaesse for CAG-EGFP plasmid preparation.
The work was supported by grants from the Centre of International Mobility of Finland, Finnish Cultural Foundation and the Academy of Finland.

1. Introduction
Creation of transgenic animals is a powerful method of investigation of gene functions in living animals1 and for unraveling disease mechanism2,3 as well as for manipulating properties of cells4. However, the procedure is rather laborious, extremely time consuming and expensive, thus warranting the use of alternative gene delivery methods such as viral injection5, neonatal ventricular injection with electroporation6 and in utero electroporation7,8. The method of postnatal non-ventricular injection and electroporation has a unique set of advantages: use of non-viral genetic constructs; possibility to create a local expression pattern in the area of interest; in situ transfection of non-migratory cell types, e.g. cortical astrocytes.
In this video, we demonstrate the detailed procedure of postnatal gene delivery to the neonatal rat brain using a plasmid encoding for the enhanced green fluorescent protein under chicken beta actin promoter with CMV enhancer (pCAG-EGFP)9. We illustrate the preparation of the plasmid-containing injection solution, manufacturing of thin glass pipettes and assembling of the microinjector on a stereotactic instrument. Then we talk about anaesthetizing rat pups with isoflurane, about performing the surgery, about the injection procedure and about the electroporation using forceps electrodes and the in vivo porator. Finally, we briefly discuss the anticipated results, perspectives and difficulties that may arise during these experiments.
2. Plasmid Preparation for Electroporation
<ol>
 <li>We usually prepare 10 &mu;l of the plasmid injection solution in a 200 &mu;l thin wall tube. This solution is sufficient for several experiments.</li>
 <li>We mix 8 &mu;l of the plasmid solution (see Materials and equipment) with 1 &mu;l of the 10X PBS (see Materials and equipment) and 1 &mu;l of 0,1% Fast Green water solution (see Materials and equipment).</li>
 <li>The final concentration of the plasmid in the injection solution should be between 1 and 3 &mu;g/&mu;l. Lower concentrations of plasmid DNA will reduce the transfection efficiency, while the higher DNA concentration will be too viscous for injection through the thin tip of the glass capillary.</li>
</ol>
3. Glass Needle Preparation
<ol>
 <li>For the glass pipette preparation, we use borosilicate glass capillary with a filament (see Materials and equipment) and a vertical electrode glass puller (see Materials and equipment) on maximum pulling and heating parameters.</li>
 <li>We then break the tip of the pipette using a piece of paper to achieve a tip 10-20 &mu;m in diameter.</li>
 <li>We check the tip diameter and shape under the microscope objective.</li>
</ol>
4. Microinjector Assembling
<ol>
 <li>Using a thin polymer capillary (see Materials and equipment) we fill approximately 1/3 of the volume of a 10 &mu;l Hamilton syringe (see Materials and equipment) with mineral oil (see Materials and equipment). Avoid air bubbles!</li>
 <li>Then we fill the glass pipette with mineral oil and insert the pipette into the Hamilton syringe. Avoid air bubbles!</li>
 <li>The syringe with glass pipette is then fixed on the microinjector connected to a controller (see Materials and equipment).</li>
 <li>The microinjection setup fixed on a stereotactic instrument (see Materials and equipment) allows us to safely immerse the tip of the glass pipette into the tube with the plasmid injection solution.</li>
 <li>When the tip touches the surface of the solution, dip it further by 1 or 2 mm and fill the pipette with approximately 1 &mu;l of the injection mix using the micro injector controller.</li>
</ol>
5. Anaesthetizing animal
All the procedures presented here were performed according to the University of Helsinki regulations for animal experiments.
<ol>
 <li>For each experiment, we fill a gas-tight syringe (see Materials and equipment) with approximately 2 ml of isoflurane (see Materials and equipment).</li>
 <li>Than the syringe is fixed on the anesthesia unit (see Materials and equipment) connected to the air flow source, the animal box and the mask fixed on stereotactic setup.</li>
 <li>On the anesthesia unit, we adjust the airflow to approximately 250 ml/min and the isoflurane level to 4 %.</li>
 <li>We place the rat pup into the animal box for 2-5 minutes.</li>
 <li>When the pup stops moving, place it on the heating pad (see Materials and equipment) attached to the stereotactic setup.</li>
 <li>Place a rostral part of pup's head into the anaesthetizing mask.</li>
 <li>It takes 5 to 10 minutes for the pup to enter the deep anesthesia.</li>
 <li>Check anesthesia deepness with a tail pinch and decrease the isoflurane inflow to about 1.5 to 2.0 %.</li>
</ol>
6. Surgery, Microinjection and Electroporation
<ol>
 <li>Treat the skin on the pup's head with the 70% ethanol.</li>
 <li>Using small scissors (see Materials and equipment) and thin forceps (see Materials and equipment), cut the skin from a scruff of the pup to its forehead.</li>
 <li>Bend skin pieces sideways and pull ear bars slightly into aural orifices of the skull for head and skin fixation.</li>
 <li>Using a binocular microscope, we locate the bregma point on the skull.</li>
 <li>Identify the region of interest using the stereotactic coordinates.</li>
 <li>Position the injection pipette above this region and mark it by dropping 25-50 nl of the injection solution onto the skull surface.</li>
 <li>Drill the skull gently and carefully at the marked point using a high speed surgical drill (see Materials and equipment) under the microscope until liquid appears in the drilled area.</li>
 <li>Fix an electropotation forceps electrodes (see Materials and equipment) on the sides of the skull with current conductive gel (see Materials and equipment) to achieve better conductivity.</li>
 <li>Remove the liquid drop from the drilled hole using small tampon (see Materials and equipment) and dip the glass needle to the hole according to the coordinates of the injection site.</li>
 <li>Infuse 25 to 100 nl of the injection solution at the rate of 5 to 20 nl/sec.</li>
 <li>Quickly remove the pipette and electroporate immediately. Electroporation is done by applying five rectangle pulses with the duration of 50 ms and amplitude of 99 V at the frequency of 1 Hz.</li>
 <li>Remove electrodes and ear bars.</li>
 <li>Sew the cut skin using a combination of a dumb (see Materials and equipment) and sharp forceps and surgical threads (see Materials and equipment).</li>
 <li>Place the pup into the warm chamber for 15-30 minutes to aid its recovery after anaesthesia.</li>
 <li>Then put the pup back to its mother's cage.</li>
</ol>
7. Representative Results
The expression of the transgene in the successfully transfected cells will appear approximately 10 hours after the electroporation, and it will remain stable for several weeks.
We microinjected the plasmid either into deep cortical layers or striatum region of postnatal day 2 (P2) Wistar rat pups and performed in vivo electroporation as described above. Brain slices (400 &mu;m in thickness) were cut after 2 to 6 days (P4-P8) using a vibrotome in cold slicing medium (Hepes buffered Earle's Balanced Salt Solution)10. Image acquisition was performed using Zeiss Axioplan 2 LSM5 Pascal confocal microscope (Zeiss, Germany) in the slicing medium at room temperature.
The injection sites in deep cortical layers and striatum contained many transfected cells expressing EGFP that were located in a compact region of approximately 200-300 &mu;m in diameter (Figure 1A, B). Within transfected cells, the EGFP expression level is sufficiently high to allow imaging of thin cellular processes (Figure 1C) including dendrites with spines of different shapes (Figure 1D) and axon bundles (Figure 1E). The robust stability of EGFP as well as cell viability after electroporation allowed tracing distal parts of axons (up to 1.5-2 mm from cell bodies) such as cortical terminals (Figure 1F).
<img src="/files/ftp_upload/2244/2244fig1.jpg" alt="Figure 1" /> Figure 1. (A) Successfully transfected cells in the striatum region of P4 rat brain delineating the plasmid injection site. (B) Transfected cells in deep cortical layers (approximately 1000 &mu;m below cortical surface) of P5 rat brain. (C) A transfected cell in the striatum region of P8 rat brain. (D) Dendritic processes of a transfected cell in the striatum region of P8 rat brain; arrows indicate different types of dendritic spines and filopodia. (E) Axons of transfected cells within collateral pathway. (F) Axons terminating at the pial surface of P8 rat cortex (arrowhead). Scale bars are 100 &mu;m on A, B, E and F; 10 &mu;m on C and D.

<ol>
	<li>Gerlai, R., Clayton, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+hippocampal+function+in+transgenic+mice:+an+ethological+perspective.+Trends+Neurosci.">Analyzing hippocampal function in transgenic mice: an ethological perspective. Trends Neurosci.</a> 22, 47-51 (1999).</li><li>McGowan, E., Eriksen, J., Hutton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+modeling+Alzheimer's+disease+in+transgenic+mice.">A decade of modeling Alzheimer's disease in transgenic mice.</a> Trends Genet. 2, 281-289 (2006).</li><li>Cryan, J. F., Holmes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ascent+of+mouse:+advances+in+modeling+human+depression+and+anxiety.">The ascent of mouse: advances in modeling human depression and anxiety.</a> Nat. Rev. Drug Discov. 4, 775-790 (2005).</li><li>Wells, T., Carter, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+neural+function+in+transgenic+rodents:+towards+a+comprehensive+strategy.">Genetic engineering of neural function in transgenic rodents: towards a comprehensive strategy.</a> J. Neurosci. Methods. 108, 111-130 (2001).</li><li>Pilpel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=reproducible+transduction+of+select+forebrain+regions+by+targeted+recombinant+virus+injection+into+the+neonatal+mouse+brain.">reproducible transduction of select forebrain regions by targeted recombinant virus injection into the neonatal mouse brain.</a> J. Neurosci. Methods. 182, 55-63 (2009).</li><li>Boutin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PLoS One. 3, e1883-e1883 (2008).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev. Biol. 240, 237-246 (2001).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat. Protoc. 1, 1552-1558 (2006).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> Proc. Natl. Acad. Sci. U S A. 101, 16-22 (2004).</li><li>De Simoni, A., Yu, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+organotypic+hippocampal+slice+cultures:+interface+method.">Preparation of organotypic hippocampal slice cultures: interface method.</a> Nat. Protoc. 1, 1439-1445 (2006).</li><li>Walantus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J. Vis. Exp. , (2007).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J. Vis. Exp. , (2007).</li><li>Umeshima, H., Hirano, T., Kengaku, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-based+nuclear+movement+occurs+independently+of+centrosome+positioning+in+migrating+neurons.">Microtubule-based nuclear movement occurs independently of centrosome positioning in migrating neurons.</a> Proc. Natl. Acad. Sci. U S A. 104, 16182-16187 (2007).</li><li>Ashwell, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of the Developing Rat Nervous System. , (2008).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (2007).</li></ol>

Methods of gene delivery into living rodent brain are well established for in utero electroporation7,8,11,12 and, more recently, for postnatal electroporation6. However, these methods are based on intraventricular injection of the plasmid DNA, which may be limiting for several applications. For example, these methods do not allow targeting cells in certain brain areas such as hippocampus, nor transfection of such non-migratory cell types as cortical astrocytes. Non-ventricular injection coupled with electroporation was first used for gene delivery into cerebellar neurons13. Our protocol illustrates the extension of non-ventricular method for other regions of rat brain. We propose that our methodological approach is a useful alternative to viral methods of gene delivery to restricted areas of interest within postnatal rat brain5.
Despite the advantages of the present method, some difficulties may be expected as discussed below.
1.Optimal age of animal
When possible, younger neonatal rat pups should be use, as a means to increase both transfection efficacy and pup survival after surgery. In our hands, the P0 pups are too small as it is difficult to target reproducibly a certain area in the brain using stereotactic coordinates obtained from the brain atlas available for this age14. In addition, the P0 pups have thin and tender skin that may impede sewing. The P3 and P4 are most convenient in terms of surgical and stereotactic manipulations as compared to P0-P2 animals, but they have hypersensitivity to isoflurane anesthesia that may cause seizures and breathing interruptions during surgery. Thus, the best option is P1-P2 rat pups which are predictable during surgery and large enough for reproducible microinjections.
2.Possible problems with microinjection
When small volumes of solution (25-100 nl) are injected through thin glass tip to the brain tissue exerts an opposing pressure, it is critical that air bubbles or leakages are carefully avoided. Failure to avoid these may cause a dramatic decrease in transfection efficiency.
3.Precision limits for stereotactic coordinates
Despite the precision of stereotactic coordinates, they have a limit at 0.3 mm reproducibility15. In our experience using animals of the same age and weight it is possible to decrease this limit to 0.2&#8722;0.1 mm which is sufficient for reproducible targeting of the CA1 hippocampal area.

<table><tbody><tr><td>2A-sa dumb Tweezers, 115mm</td><td>equipment</td><td>XYtronic</td><td>XY-2A-SA</td><td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td></tr><tr><td>Biological Temperature Controller with stainless steel heating pad</td><td>equipment</td><td>Supertech</td><td>TMP-5b</td><td></td></tr><tr><td>Borosilicate tube with filament</td><td>material</td><td>Sutter Instrument Co.</td><td>BF120-69-10</td><td>Glass needle</td></tr><tr><td>Disposable drills</td><td>material</td><td>Meisinger</td><td>HP 310 104 001 001 008</td><td></td></tr><tr><td>Dulbeco&amp;rsquo;s PBS 10X</td><td>reagent</td><td>Sigma-Aldrich</td><td>D1408</td><td></td></tr><tr><td>Dumont #5 forceps, 110 mm</td><td>equipment</td><td>Fine Science Tools</td><td>91150-20</td><td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td></tr><tr><td>Ealing micr&amp;#8211;lectrode puller</td><td>equipment</td><td>Ealing</td><td>50-2013</td><td>Vertical electrode glass puller</td></tr><tr><td>Ethilon monofil polyamide 6-0 FS-3 16 mm 3/8c</td><td>material</td><td>Johnson &amp;amp; Johnson</td><td>EH7177H</td><td>Surgical threads</td></tr><tr><td>Exmire micro syringe 10.0 ml</td><td>equipment</td><td>Exmire</td><td>MS*GLLX00</td><td>Gas-tight syringe</td></tr><tr><td>Fast Green</td><td>reagent</td><td>Sigma-Aldrich</td><td>F7252</td><td></td></tr><tr><td>Forceps electrodes</td><td>equipment</td><td>BEX</td><td>LF650P3</td><td>Treat with 70% ethanol for disinfection prior to use</td></tr><tr><td>Foredom drill control</td><td>equipment</td><td>Foredom</td><td>FM3545</td><td>Surgical drill power supply and control. Currently available analogue is micromotor kit K.1070 (Foredom) </td></tr><tr><td>Foredom micro motor handpiece</td><td>equipment</td><td>Foredom</td><td>MH-145</td><td>Currently available analogue is micromotor kit K.1070 (Foredom)</td></tr><tr><td>Gas anesthesia platform for mice</td><td>equipment</td><td>Stoelting Co.</td><td>50264</td><td>Assembled on stereotaxic instrument</td></tr><tr><td>Isoflurane</td><td>reagent</td><td>Baxter Internationl Inc.</td><td>FDG9623</td><td></td></tr><tr><td>Micro dressing forceps, 105 mm</td><td>equipment</td><td>Aesculap</td><td>BD302R</td><td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td></tr><tr><td>Microfil</td><td>material</td><td>World Precision Instruments, Inc.</td><td>MF34G-5</td><td>Micro syringe filling capillaries</td></tr><tr><td>Mineral oil</td><td>reagent</td><td>Sigma-Aldrich</td><td>M8410</td><td></td></tr><tr><td>NanoFil Syringe 10 microliter</td><td>equipment</td><td>World Precision Instruments, Inc.</td><td>NANOFIL</td><td>Hamilton syringe</td></tr><tr><td>plasmid CAG-EGFP</td><td>reagent</td><td></td><td></td><td>Extracted and purified with EndoFree Plasmid Maxi Kit (Qiagen) and dissolved in nuclease free water to concentration 1.5 mg/ml</td></tr><tr><td>Pulse generator CUY21Vivo-SQ</td><td>equipment</td><td>BEX</td><td>CUY21Vivo-SQ</td><td></td></tr><tr><td>Schiller electrode gel</td><td>reagent</td><td>Schiller AG</td><td>2.158000</td><td>Conductive gel</td></tr><tr><td>Small animal stereotaxic instrument</td><td>equipment</td><td>David Kopf Instruments</td><td>900</td><td></td></tr><tr><td>St&amp;#8211;lting mouse and neonatal rat adaptor</td><td>equipment</td><td>Stoelting Co.</td><td>51625</td><td>Assembled on stereotaxic instrument.Treat earbars with 70% ethanol for disinfection before use in surgical manipulations</td></tr><tr><td>Student iris scissors, straight 11.5 cm</td><td>equipment</td><td>Fine Science Tools</td><td>91460-11</td><td>Treat with 70% ethanol for disinfection before use in surgical manipulations</td></tr><tr><td>Sugi absorbent swabs 17 x 8 mm</td><td>material</td><td>Kettenbach</td><td>31602</td><td>Surgical tampons</td></tr><tr><td>UMP3 microsyringe pump and Micro 4 microsyringe pump controller</td><td>equipment</td><td>World Precision Instruments, Inc.</td><td>UMP3-1</td><td>Microinjector and controller</td></tr><tr><td>Univentor 400 Anesthesia Unit</td><td>equipment</td><td>Univentor</td><td>8323001</td><td></td></tr></tbody></table>

gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

Watch this Scientific Journal Video about Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation at JoVE.com

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

gene-delivery-to-postnatal-rat-brain-non-ventricular-plasmid

Nanyang Technological University

Research

JoVE Journal

Neuroscience

15.0K Views.  University of Helsinki. This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Video: Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

Biology

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 ...

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.

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.

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

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...

Developmental Biology

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

A Comprehensive Protocol for Delivering Intrathecal Injections in Neonatal Mice

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

Author Spotlight: Intrathecal Injection &#8211; An Efficient and Reliable Delivery Method to Test the Efficacy of Gene Editing in Neonatal Mouse Brains

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

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Neonatal Subventricular Zone Electroporation

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Neonatal Pial Surface Electroporation

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation