NeuroPorator: An open-source, current-limited electroporator for safe in utero gene transfer

Background: Electroporation is an effective technique for genetic manipulation of cells, both in vitro and in vivo . In utero electroporation (IUE) is a special case, which represents a fine application of this technique to genetically modify specific tissues of embryos during prenatal development. Commercially available electroporators are expensive and not fully customizable. We have designed and produced an inexpensive, open-design


Introduction
Genetic manipulation of animals has brought unprecedented possibilities to model human diseases in vivo.Animal models, especially mice and rats, have been modified with various genes up to entire artificial transgene cassettes inserted to their genomes.Such lines of genetically modified animals usually carry germinal alteration, e.g.mutation, meaning the gene modification is present in all cells of the model animal.Many of these animal models have been successfully developed and established in preclinical research of a wide spectrum of diseases.However, it is increasingly recognized that the pathogenesis of various disorders can be attributed to somatic mutations occurring during early stages of embryonal development.The mutation then affects a limited number of cells (daughter cells), resulting in tissue mosaicism, affecting multiple parts of the organ.The specific gene, character, and embryonal timing of the mutation determine the severity of the phenotype.In neuroscience, recent genetic studies demonstrated that somatic mutations occurring during brain development are associated with various neurological conditions, such as malformations of the cortex (Lim et al., 2015) or brain tumors (Zhang et al., 2021), which are often related to epilepsy.
In contrast to breeding mouse lines with germinal mutations, where the mutation is hereditary to the offspring, modeling brain disorders due to somatic mutations require introducing the mutation in the naïve developing brain to mimic a spontaneous mutation occurring during the gestational period in humans.
Affecting a specific population of cells in a developing embryo can be achieved by a technique of in utero electroporation (IUE).The goal of electroporation is to deliver DNA into cells using an electric field.Transient electric fields form pores in the cell membrane, allowing charged DNA molecules to enter the cell.Electroporation was first introduced more than 40 years ago for transfecting mouse cell lines in vitro (Neumann et al., 1982;Potter et al., 1984).The technique had been modified for in vivo usage (Saito and Nakatsuji, 2001;Tabata and Nakajima, 2001) and successfully applied to study neuronal migration and various aberrations of brain development (Ackman et al., 2009;Bogoyevitch et al., 2012;Feliciano et al., 2011;Lim et al., 2015;Tsai et al., 2005).
In terms of special equipment, IUE requires platinum tweezer electrodes and a device that can generate and deliver a series of short electric pulsesmostly termed as electroporator.While both tools are commercially available, the electroporator comes at a substantially high price that can prevent many laboratories from taking advantage of the IUE method.Here, we present an alternative termed as NeuroPorator: A fully documented, open-design, easy-to-make electroporator that is affordable, largely configurable, and fits the parameters for IUE.
Our electroporator design comprises a current-limiting circuit to protect the embryos.Additionally, it includes a built-in data acquisition (DAQ) module that enables for real-time visualization and recording of the actual voltage and current applied during IUE to each embryo.
NeuroPorator can be assembled within two days with only basic knowledge of soldering and almost no necessary knowledge of programming.In Supplementary materials, we have attached complete documentation with a detailed description of the manufacturing process, bill of materials, 3D models of all printable parts, documented and customizable Arduino code, and a parts layout for a printed circuit board (PCB) or solderless assembly.We have demonstrated routine usage of NeuroPorator in modeling a specific neurological disorder caused by localized somatic mutationfocal cortical dysplasia (FCD), which often manifests with epileptic seizures.

NeuroPorator design and hardware
NeuroPorator consists of three main parts -Pulse generator, Current limiter, and Pulse recorder modules.The source of energy in our design consists of five 9 V batteries connected in series to create a 45 V voltage source.Voltages of 30 V-45 V can be found as typical in literature for IUE (Baumgart and Baumgart, 2016;Saito and Nakatsuji, 2001;Tabata and Nakajima, 2001).
The Pulse generator module is based on an Arduino Uno Rev3 microcontroller (Arduino LLC, Italy) and a fast reed relay (Fig. 1(a)).The fast reed relay can be directly driven by an Arduino digital output (+5 V).The module produces precisely timed pulse trains.The Arduino board continuously senses a contact on the sustain pedal to trigger the predefined pulse sequence.This is sent to both the reed relay and a small active buzzer (Fig. 1(b)) for audio signaling of the pulse train.We provide a ready-to-use code sample for the microcontroller, including the default settings (five 50 ms pulses, 950 ms inter-pulse intervals), in the Supplementary material (Default_code.ino).The code was modified and uploaded to the board using Arduino Integrated Development Environment, Arduino IDE 2.0.The Pulse generator module connects voltage supply with the Current limiter module (Fig. 1(c)).
The Current limiter serves as a module that dynamically adjusts its output voltage to keep output current below a predefined value.It includes one additional fast relay (G5V-1-DC24, Omron, Japan) that connects the pulses (NO pin of the relay) to the output during the high phase and short-circuits the output (the electrode contacts) during the low phase.The rated voltage of the relay is 24 V, but up to 48 V driving voltage is tolerated by the relay.
The current limitation mechanism is set by a combination of two transistors (T1, T2) and a potentiometer (P1).Transistor T1 is open when T2 is closed, as the voltage on T1 base is high.The voltage at the base of T2 is given by the product of P1 resistance and the current through P1 that in series flows through the electrode.Once the electrode current rises, the T2 base voltage also rises, crosses the threshold (typically 0.6-0.7 V), and T2 opens.Once T2 opens, the voltage on T1 base drops as it is given by power supply voltage divided by R2 resistance (high) and T2 collector-emitter resistance (low for open transistor).T1 then closes.This mechanism sets this circuit to a steady state with the maximum current through T1 (and in series also through the electrode), governed by the P1 resistance as their product is kept under the T2 base threshold voltage.P1 resistance is adjustable (blue multiturn trimmer in Fig. 1(d)) to enable the setting of a maximum current.We incorporated a fast bistable relay that opens during the pulse and shortcircuits the electrode pins between pulses to minimize capacitancerelated artifacts, which would distort the pulse shape.Resistor R1 serves as a circuit protection and concurrently as a load of known resistance.The applied current can be monitored and calculated from the voltage (SenseCurrent+, SenseCurrent-) across R1.Resistors R4 and R3 constitute a voltage divider of substantially higher total resistance compared to the expected impedance at the electrode.The actual pulse voltage and shape is read out across R3 (SenseVoltage+, SenseVoltage-).Unwanted back-induction voltages generated at the relay coil during pulse ending are eliminated by a Zener diode (ZD).We provide three variants of PCB layouts made from off-the-shelf prototyping boards in the Supplementary materials (Current limiter PCB layouts.pdf).
The aforementioned modules are accompanied by a third one-Pulse recorder-for which we use a commercially available system, USB-6001 (National Instruments, USA).It mainly serves to monitor and record the timing and amplitude of the pulses using a PC application (e.g.DAQExpress, National Instruments, USA).The module is connected to the Current limiter output through the SenseCurrent and SenseVoltage wires (Fig. 2).
The Pulse generator and the Current limiter modules were assembled with solder and hot glue.Boxes for both modules and the main box were 3D printed with an FFF printer (Prusa i3 MK3S+, Prusa, Czech Republic).The 3D object files are attached as Supplementary materials in.stl and.step formats.Detailed assembly and setting instructions for all modules, connecting cables, and PCB variants are given in the Supplementary text and figures as well as the list of all necessary electronic parts (bill of material -NeuroPorator_BOM.xlsx).The whole process of assembling NeuroPorator is thoroughly described in Supplementary materials: Assembly_instructions.pdf.

Animals
Mice used in this study were crossbreeds of an SST-IRES-Cre strain (Jackson Laboratory cat.number #018973), a PV-P2A-Cre strain (Jackson Laboratory, USA, cat.number #012358), or a VIP-IRES-Cre strain (Jackson Laboratory cat.number #010908) and a flex-tdTomato reporter strain (Jackson Laboratory cat.number #007909).All strains were on a C57Bl/6 J background except VIP-IRES-Cre strain, that was based on a mixed C57BL/6×129S4Sv background.Mice were kept under standard conditions in a room with controlled temperature (22±1 • C) and a 12/12 h light/dark cycle and ad libitum access to food and water.

In utero electroporation
Pregnant mice (day 14.5±0.5 post-fertilization) were anesthetized with isoflurane (5% induction, 1.5% maintenance).The fur on the abdomen was shaved off and a 1.5 cm incision was made in the middle of the abdomen to expose the muscle layer.A 1 cm-long laparotomy was performed in parallel with the linea alba to access the uterus.The exposed uterine horns were then placed on sterile gauze around the incision and frequently moistened with warm phosphate-buffered saline (PBS).To induce FCD and mark the transfection localization, embryos were injected with a plasmid mixture containing 3 µg/µl of expression plasmid CAG-mTOR 7280 T>C -IRES-EGFP (SOVARGEN, Korea [1]), or its wild-type variant together with 1.5 µg/µl of expression plasmid CAG-EGFP, or CAG-iRFP (infrared fluorescent protein).Animals in one control group were electroporated with 1.5 µg/µl CAG-EGFP only.The expression plasmids were diluted in 2 ug/ml Fast Green (F7252, Sigma, USA) for visualization.Each embryo was injected into the left lateral ventricle using a pulled and beveled glass capillary (Fig. 3(a)).After each injection, forceps-type electrodes (3 mm, CUY650P3, Nepagene) were positioned on the head of the embryo, with the positive electrode facing the left side of the embryo's head, and electroporation of the plasmids was performed using NeuroPorator that delivered five 50 ms pulses with 950 ms inter-pulse intervals (Fig. 3(b)).
The applied voltage was 30-45 V (dynamically adjusted by the device) and the current limitation was set to 40 mA.

Electrode implantation and EEG recording
To evaluate the epilepsy phenotype induced by FCD, successfully electroporated pups (confirmed by fluorescence screening) were fostered and later implanted with recording electrodes and placed into a video-EEG monitoring chamber.Custom EEG connectors were prepared by soldering PFA-coated silver wires (AM Systems, Inc., USA, cat.no.786000) to prefabricated connectors (TME Electronic Components, Poland, cat.no.DS1065-03-2*6S8BV).Animals were implanted with EEG electrodes at 8-10 weeks of age.The animals were anesthetized with isoflurane and their heads were fixed by a mouse gas anesthesia head holder (Kopf Instruments, USA, cat.no.923-B).After exposing the dorsal skull, the exact position of GFP signal was assessed using a green bandpass filter (525/45 nm, Edmund Optics) attached to the surgical microscope and a 488 nm laser pointer (Sanwu, PRC).Using a highspeed microdrill (Osada Electric Co., Ltd., Japan, cat.no.OS-40) with 0.3 mm drilling bits, 2-6 holes were drilled through the skull carefully to avoid any damage to dura mater.At least one electrode was positioned directly over the FCD and one electrode to the mirror position in the contralateral skull area.Two holes were positioned over the cerebellum for grounding/reference electrodes.All electrode bare endings were placed on the surface of the dura, and the holes were covered with bone wax (SMI, Belgium, cat.no.Z046).Electrodes and the connector were attached to the skull using cyanoacrylate glue gel (Loctite, USA, cat.no.1363589) accelerated with cyanoacrylate glue accelerator (Bob Smith Industries Inc., USA, cat.no.BSI-152).
After the surgery, animals were allowed to recover for five days and subsequently placed into the video-EEG recording chambers.All mice were individually video-EEG monitored for four weeks for 24 hours a day.Recorded electrographic activity was amplified, bandpass filtered (0.1 Hz-1.6 kHz), and digitized at 5 kHz using a 32-channel amplifier chip (Intan Technologies, USA, cat.no.RHD2132).Finally, EEG data were manually labeled for seizures using custom-made software in MATLAB 2019b (Mathworks Inc., USA).

Tissue clearing and microscopy
Animals were overdosed with a ketamine/xylazine intramuscular injection (120 mg/kg and 20 mg/kg, respectively) and intracardially perfused with cold saline followed by 4% paraformaldehyde in PBS.Brains were explanted and postfixed overnight in 4% paraformaldehyde, after which they were stored in 0.05% sodium azide in PBS until they were processed according to the CUBIC clearing protocol (Susaki et al., 2015) for subsequent morphological imaging.
Images were taken using a custom-made two-photon microscope with a femtosecond laser (Chameleon Vision S, Coherent), an 8 kHz resonant scanner (LSK-GR08, Thorlabs), a 20x XLUMPLFLN waterimmersion objective (Olympus), and two silicon photomultipliers (Hamamatsu).Cytoarchitecture of the fluorescently labeled tissue was recorded using sequential two-photon tomography (Ragan et al., 2012).In this procedure, a block of cleared tissue is embedded in agarose and glued to a vibratome inset.The brain was cut at a specific transversal position and moved to the two-photon microscope.The coronal section of the brain was mapped for fluorescence and tiled with an appropriate number of 800 µm-sided fields of view.At each field of view, the microscope took a fine 1200 µm-thick Z-stack, 2 µm step in Z, with 800 ×800 µm (2048 ×2048 pixels) X-Y field of view.

Improved design to minimize the tissue damage
The current between the tweezer electrodes can flow through many ways (Fig. 4(a)).If the embryo is not positioned properly and the electrode is not compressed against the uterine wall, instead of the embryo's brain tissue, a large proportion of the current can flow through approximately four times more electrically conductive amniotic fluid (Pachi et al., 2001).However, all the current must flow through the uterine wall-electrode contacts.If an electroporator works only as a pulsed voltage source, the current flowing between the contacts of the electrode is inversely proportional to the impedance of the sample, but is not limited (Fig. 4(b), yellow).We incorporated current limiting circuitry to NeuroPorator to protect the uterus and embryos from excessive current (Fig. 4(b), green).

N. Procházková et al.
To assess the performance and safety of NeuroPorator, we have recorded the voltage and current delivered to mock samples of known resistance.We used resistors with values in range we observed during IUE.NeuroPorator was set to limit the current at 40 mA.Fig. 5(a) shows the voltage and current recordings of pulses applied to 600 Ohm, 900 Ohm, and 1200 Ohm, respectively.Duration of pulses was set to 50 ms and the precise onset and offset edges of the pulses can be seen from the two zoomed pulses for different impedances (Fig. 5(a), inset).We measured and plotted the output voltage and current in Fig. 5(b) for resistors of 11 different values.For samples of high impedance (1200 Ohm and higher), the behavior of NeuroPorator is approximately Ohmic as the preset current limit cannot be reached and maximum voltage of the source is delivered.For lower impedances, e.g., 900 Ohm, 600 Ohm, NeuroPorator adjusts the output voltage to limit the current to the preset value.

Comparison to existing electroporators
The parameters and features of NeuroPorator compared to commercially available electroporators and previously published custom-made electroporators are listed in Table 1.
NeuroPorator delivers rectangular unipolar pulses.This is also the case for most of the compared devices; the only one with variable bipolar pulses is NEPA21 (Nepagene).The pulse timing of NeuroPorator is highly variable with comparable time precision to those declared by other electroporators.Some electroporators can deliver voltages for both in utero and in vitro electroporation (ECM830 by BTX), others are focused only on in vitro application (Portoporator©), and finally, there are electroporators with voltages primarily complying with the ranges for IUE, including NeuroPorator.The prominent advantages of Neuro-Porator, which are absent in the compared devices, are the fully adjustable current limitation and the real-time displaying and recording of the voltage and current curves.Furthermore, it is possible to assemble the device in only two days and all necessary parts cost approximately $550.

Proof of NeuroPorator efficacy
NeuroPorator has been thoroughly tested in our laboratory for IUE of a plasmid-containing mutated mechanistic target of rapamycin (mTOR) gene to induce FCD-a very common cortical malformation (Crino, 2015).A specific point mutation (7280 T>C) leads to a constitutively active protein and alters neuronal migration, growth, and proliferation [1].Normally developed neocortex has a stereotypical cytoarchitecture, layered structure, and clear margins between the gray matter, consisting of cell bodies, and the white matter, made up of myelinated axons (Fig. 6  (a)).If a plasmid (Fig. 6(a), top) is injected into a brain lateral ventricle in mouse embryos and electroporated at day E14.5, the plasmid primarily enters radial glia and newly formed neurons that should migrate into the upper layers of the cortex (layer 2/3 in mice).Plasmid containing only a control fluorescent protein EGFP (Fig. 6(a), left) leads to fluorescence-labeled layer 2/3 of the cortex (Fig. 6(b)).However, disrupting the mTOR signaling pathway using electroporation of mutated mTOR gene at day E14.5 leads to a dysplastic cortex with disrupted laminar structure, absence of clear borders between gray and white matter, ectopic neurons (Fig. 6(a), right, Fig. 6(c)), and abnormal neuronal phenotypes.These neurons (Fig. 6(d)) are characterized by enlarged somata, atypical dendritic branching, and abnormal connectivity.The FCD displays high endogenous epileptogenicity characterized by repeated spontaneous epileptic seizures (Fig. 6(e)).
Images and traces shown in Fig. 6 are based on the groups of mice prepared by Experimenter C (Fig. 7).The yield of the chosen FCD model of chronic epilepsy is relatively low.Out of 24 mice that were in utero electroporated with the mutated mTOR gene and that were later preselected based on their relatively large lesion, only 9 developed epileptic phenotype and spontaneous seizures captured at video-EEG monitoring (37.5%).None of the mice electroporated with sole EGFP plasmid showed any signs of epileptic activity.
We also evaluated NeuroPorator in terms of survival rate and success rate in the hands of three independent, experienced experimenters working on separate projects (Fig. 7).
We evaluated mean numbers of embryos found in each pregnant mouse, mean electroporated embryos, number of pups found one day after birth, and number of pups with a clearly observable fluorescent spot.Experimenters A and B mostly skipped the two embryos closest to the cervix.This is usually recommended to prevent complete abortion that can occur if those two embryos are damaged.Experimenter C followed such recommendation only partially.Mothers after the electroporation died only rarely.Out of the 70 mice described in Fig. 7, only two (<3%) mice died before or after the delivery.Experimenters A and C worked with mice on C57Bl/6 J background.Experimenter B worked with crosses that were also partially based on 129S4Sv background; embryos/pups had one fourth of 129S4Sv background and mothers were either fully based on C57Bl/6j background (half of them) or were of mixed background C57BL/6×129S4Sv.Although many variables cannot be separated, our data show that while using NeuroPorator, the mean success rate across various experimenters and different animal groups can be as high as ~40%.

Discussion
In this publication, we present a home-built electroporator, Neuro-Porator, demonstrate its properties, and verify its functionality by inducing FCD through IUE.The main advantages of our proposed solution are safety, data recording, low price, adjustability, and an open design of the electroporator.In this section we discuss each of them in Fig. 5. of NeuroPorator: (a) Voltage and current pulses recorded at the testing load; the load consisted of 600 Ohm, 900 Ohm and 1200 Ohm resistors, respectively.The current limit was set to 40 mA.One pulse for a 600 Ohm load and one pulse for a 1200 Ohm load are zoomed in the inset.With low load impedance, the electrode voltage is being limited; with high impedance, the output voltage reaches the maximum voltage NeuroPorator can deliver.(b) Current limiting performance of NeuroPorator shown as the maximum recorded current and voltage pulses for various load values.The current limiter action can be seen from resistor samples of 1000 Ohm and below.comparison to available products.

Safety
A successful electroporation of embryonal brain cells relies on creating the correct conditions for DNA to enter the cells.The strength of the pulsing field needs to be sufficient to create the pores in the membrane.However, the delivered energy must be minimized to prevent the tissue from heat and damage.The electrode is apposed directly to the uterine wall surrounding the head of the embryo (Fig. 3(b)).In case of a loose arrangement of the electrodes, uterus, and embryo (see Fig. 4(a)), which often happens, the resistance of the sample between electrodes can drop dramatically.If the voltage is kept constant in this case, the applied electric current increases.According to our experience, this can damage the uterine wall and lead to the complete abortion of all embryos.We added easily adjustable current limitation to protect embryos.
The commercially available electroporators are operated with chosen voltages, but the output current is not properly limited and is usually  simply given by Ohm's law.Balancing all parameters of the procedure is difficult, since one of the crucial parameters, along with the mechanical manipulation with the uterus, is the sample (wrapped embryo) impedance.To prevent the excessive currents flowing through the uterine wall (yellow arrow in Fig. 4(b)), we applied an analogue current limiter to the output circuit of NeuroPorator.This limiter bends the current-voltage (I-V) curve by dynamically adjusting the voltage and ensures that the maximum current does not exceed the chosen value (green arrow).
Constraining both applied voltage and current protects the uterus and embryo from harmful insults.The result in individual embryos can be either optimal or suboptimal and ineffective transfection, but not a complete abortion of all embryos due to uterine damage.It is very difficult to exactly quantify the added safety and success rates in comparison with other groups.First, almost no groups publish such numbers.Second, a vast number of variables are involved.The success rate inter alia strongly depends on the experience level of the experimenterwhether the embryos are gripped enough, but not too much, whether the surgery is quick, whether the embryos are kept warm, whether the capillary is sharp and not broken, and other factors.Also, the mouse strain plays a big role.We mostly work with C57Bl/6 Jbased transgenic mouse strain and the presented electroporator was also evaluated in this strain.However, C57Bl/6 J litter count is relatively small and C57Bl/6 J females are known to not be very good mothers (Rennie et al., 2012).We showed that when using NeuroPorator, we achieved average success rate around 40%.As we also involved the pups that were probably eaten by their mothers and pups found dead because the absence of their mothers' lactation, the number would be probably substantially higher if we evaluated NeuroPorator in an outbred strain such as CD1 white mice.

Data recording and visualization
Direct visualization of the voltage and the current of the ongoing pulse trains is important for instant feedback and enables direct control of the procedure.It also facilitates learning of the procedure for lab workers starting with the method.Although commercially available electroporators display current and voltage values in real time, Neuro-Porator can record them throughout the whole operation.This feature allows the user to look back on each pulse train and relate them to IUE output.

Price and open design
Commercially available electroporators are debugged and reliable, however, they are also relatively expensive, most of them exceeding $10k in cost.In comparison, all material for assembling our device can be purchased for approximately $550.With the instructions attached in the Supplement, it can be assembled in two days with very basic equipment and soldering skills.
Higher price is the model case for the philosophy of Open Labware (Baden et al., 2015), that aims at making advanced laboratory devices more affordable.Recently, there were published two well-documented and designed custom-made open-source electroporators (Bullmann et al., 2015;Schmitt et al., 2019).However, the first of them, called Portoporator, is designed for electroporation of cells in cuvettes and thus operating at higher voltages.The second one (Bullmann et al., 2015) is designed directly for IUE and was shown to work properly.However, its versatility is somewhat limited, e.g., it does not enable pulse trains.
Our design offers high pulse train adaptability achievable with only minor adjustments of parameters in the provided code sample.Versatility of the code brings sub-millisecond time resolution, variable number and duration of the pulses, and variable inter-pulse intervals.The presented design can be easily further modified.With specific positioning of the bipolar electrode, the DNA can be targeted to selected brain areas such as cortex, hippocampus, striatum, and others (Kolk et al., 2011;Wang et al., 2012).This spectrum of targetable structures has been substantially broadened by introduction of tripolar electroporation (dal Maschio et al., 2012).NeuroPorator can be used as a source for tripolar electroporation with a proper external wiring of the electrodes.
In practice, we connect the Arduino board via a USB connector for both programming and power.However, it can be also powered from a sixth 9 V battery inside the NeuroPorator box, rendering the device independent of a power supply.Another modification can be done by altering the visualization module.If one does not need the real-time visualization and recording of the actually applied pulses, the data acquisition system can be disconnected and NeuroPorator can be fully independent and portable.The data acquisition system USB-6001 Fig. 7. Evaluation of the survival rate and success rate of IUE using NeuroPorator by three different experimenters.In three separate projects, plasmids carrying a gene for two variants of mTOR, either wild-type (mTOR wt ) or mutated (mTOR mut ), were electroporated together with a plasmid carrying a gene for a fluorescent protein EGFP or iRFP.In one group, Experimenter C electroporated only plasmid with a gene for EGFP.The numbers below each graph denote the number of electroporated pregnant mice for the respective projects.For each group, mean fractions of successfully electroporated, viable pups, and electroporated embryos are shown.Error bars represent standard error of the mean (SEM).(National Instruments) is not too expensive, but if one wants to reduce the price of NeuroPorator even more, USB-6001 can be omitted.This would reduce the price down to ~$150.Alternatively, it is possible to design and add a visualization module to the device or an SD-card shield for data saving (e.g.Assembled Data Logging shield for Arduino for $15; Adafruit).

Conclusion
Here, we presented a fully documented open-design electroporator specially developed for in utero electroporation-NeuroPorator.Within the philosophy of Open Labware (Baden et al., 2015), the construction of NeuroPorator is easy and the material cost is low.We believe that by using very detailed documentation and assembly instructions, many laboratories will start using this device.NeuroPorator makes the technique of in utero electroporation more affordable and easier to implement.

Fig. 1 .
Fig. 1. Circuit schemes and arrangement of the pulse-delivering NeuroPorator modules: (a) Circuitry of the Pulse generator module showing a fast reed relay driven by the Arduino Uno R3 digital pins.(b) Arrangement of the Pulse generator module in a 3D-printed case.(c) Circuitry of the Current limiter module with described input output points.(d) Arrangement of the Current limiter module.

Fig. 2 .
Fig. 2. Arrangement of the NeuroPorator modules: (a) Block diagram of NeuroPorator, including an Arduino-based Pulse generator, analogue Current limiter, and a commercially available Pulse recorder (National Instruments USB-6001).The pulses are energized from five 9 V batteries connected in series.(b) Arrangement of individual modules inside the NeuroPorator box; here, the Pulse generator module has been removed.(c) Overall look of the presented electroporator together with CUY650P3 tweezer electrodes.

Fig. 3 .
Fig. 3. Basic principle of gene delivery through electroporation with focus on in utero conditions: (a) Outline of in utero electroporation of the brain.Each embryo is gently positioned in the uterus, a small volume of DNA solution is injected into embryo's brain ventricle through the uterine wall, and tweezers with platinum-plated electrodes are attached to the uterine wall, surrounding the embryo's head.Electrical pulses to transduce the targeted cells are delivered.(b) Real image of electroporation of previously injected embryo.The injected plasmid is visible as dark green substance in the lateral ventricle.

Fig. 4 .
Fig. 4. Reasoning for optimal parameters for safe and effective IUE: (a) Drawing of an embryo's head and electric current streamlines.Arrowheads are oriented in the direction of expected DNA movement.Current is induced by the electrode attached to the uterine wall.(b) Function principle of the current limiting feature of our electroporator in a current-voltage characteristic.Green and yellow curves are functions of one parameter-the impedance between contacts of the electrode (load).With a constant voltage (30-45 V)-delivering electroporator, voltage-current combinations can be found as points in a certain area, here depicted as a dashed line-rounded rectangle, depending on the load impedance.The yellow line represents a function of decreasing impedance for 45 V.With lower impedance, the current can easily reach harmful values.The Current limiter module of NeuroPorator does not let the curve continue straight and dynamically limits the voltage on the electrode to ensure that the maximum allowed current is not exceeded (green curve).

Fig. 6 .
Fig. 6.Performance of NeuroPorator in real mouse cortex: (a) Plasmids that were electroporated at day E14.5 included either a gene for constitutively active mTOR and a gene for enhanced green fluorescent protein or the fluorescent protein gene solely.Illustration of normal cortex with its stereotypical cytoarchitecture and layered structure (a, bottom left) and dysplastic cortex with smeared lamination and ectopic dysmorphic neurons (a, bottom right).(b) The cytoarchitecture of the cortex with almost all fluorescently labeled neurons migrated into layer2/3 after IUE with a control plasmid.Dysplastic cortex with fluorescently labeled neurons throughout the gray and even the white matter after IUE with a plasmid carrying the mutated mTOR gene variant.(d) Dysmorphic neurons with atypical dendritic branching and highly enlarged somata.E) Local field potential recording from the dysplastic cortex during an epileptic seizure.Legend: Scale bars correspond to 500 µm in (b), (c) and 50 µm in (d).WM = white matter.In (B), (C) white dashed lines depict the cortical surface and the gray matter / white matter border.Blue lines represent the approximate borders of layer 2/3 of the mouse somatosensory cortex.

Table 1
Comparison of the parameters between NeuroPorator, the commercially available electroporators, and the previously published custom-made electroporators.Abbreviations: HV, high-voltage; LV, low-voltage.