Application Support – NEPA21

Programming the NEPA21 is easier than it looks

Here’s how to program the NEPA21 system: NEPA21 Programming Guide

Do you own a NEPA21 system? Get a for user manual with more details.

Why is it important to record electrical output from each pulsing set tested?

The NEPA21 does not deliver constant current; instead it delivers constant voltage under controlled electric resistance. The current delivered is displayed on the NEPA21. This method allows for control of all three variables: voltage, current and resistance.  These three electrical parameters, not just current, are crucial for the best results.

We always recommend that users record ALL output electrical parameters when first performing a new application. These values include: Impedance (kΩ), Voltage (V), Current (A), and Energy (J). Impedance is another measure of electrical resistance under an AC trickle check.

Once optimal conditions are established, you should ALWAYS CHECK IMPEDANCE just prior to pulsing. If it falls outside of the recommended range, the volume or ionic conditions needs to be adjusted. If the Impedance value is in the correct range proceed with pulsing immediately.

With this method you can set both Voltage and Impedance, so that Current and Energy are highly defined and reproducible. Ohm’s Law: I = V/R

When optimizing pulsing conditions, electrical output can often be correlated with results. For example, lower energy delivered (J) often results in higher cell viability. Conversely, higher energy delivered (J) often results in higher transfection efficiency. Finding the balance between healthy cells and efficient delivery is the goal of optimization; once a particular protocol is optimized for an application or cell type, future optimization is rarely needed.

Cuvette Electroporation Method

The NEPA21 is most commonly used with electroporation cuvettes. We have an in-house written protocol available to any lab that owns a NEPA21 system. Please ask us for this document.

The NEPA21 cuvette protocols usually use 3- or 4-phases of electrical pulsing, including a Poring Pulse Phase to “open holes” in the membrane, followed by a Transfer Pulse Phase to move the charged molecules (usually DNA, RNA or proteins) into the cell. Both phases are essentia for high cell viability and transfection efficiency.

Advantages:

• No requirement for special buffers or expensive reagents
• Fully adjustable electrical pulsing parameters
• Non-capacitor design for handling physiological salt conditions
• Can be used to transfect large plasmids (10+ kb)
• Works with proteins, RNA, and gene editing complexes
• Proven on hundreds of cell lines and cell types.

Key publications: The NEPA21 has been referenced in more than 800 publications since 2012. Most of these use 2 mm electroporation cuvettes. We would be happy to help you find the most pertinent publications for your lab’s particular experimental focus.

Using the NEPA21 cuvette method:

Ask us for both a written protocol and for our suggested pulsing parameters for your lab’s cells of interest. These can be supplied to any NEPA21 lab.

Cell Types:

Hundreds of cell lines and cell types have been demonstrated to work with the NEPA21 system. Below is a quick summary: click here for an up-to-date list of dozens of cell types with images and expected results.

• Primary cells (including stem cells, neurons, and many more)
• Cell lines (all common and many obscure)
• Immune cells (including B and T lymphocytes)
• Cell clusters (including organoids and dissociated cells from tissues)
• Walled cells (including Chlamydomonas and Diatoms)
• Marine protists (e.g., Euglena)

Electrode Types:

CU500 Electroporation Cuvette Chamber

• Accepts industry-standard electroporation cuvettes
• Ask about samples of 2 mm gap electroporation cuvettes here.

2 mm gap Electroporation Cuvette

• 100 μL volume
• buffer type: Opti-MEM
• most commonly recommended
• 1 x 10⁶ cells
• 10 μg of plasmid DNA
• target Impedance reading is 30 Ω to 55 Ω

Tips for Success:

1. Please ask us for a spreadsheet with recommended, field-tested, parameter sets to try. When first optimizing a new cell line or cell type, use ALL of the recommended pulsing parameters for that cell type.
2. Always record output electrical data – especially the IMPEDANCE value. We are happy to offer suggestions for further optimization, but we can only help you interpret your results when the electrical data is available.
3. We recommend performing the transfection in serum-free (or at minimum serum-reduced) Opti-MEM. The cells should be washed at least twice to ensure good electric-field formation.
4. Use a high quality source for reagents. In the case of DNA plasmids purify using an endotoxin-free kit. For gene editing reagents we recommend using the Cas9 protein instead of mRNA.
5. If you are using your own plasmid vector, please ensure that it has already been fully qualified and works for other transfection methods. Otherwise, ask us about our control DNA, which contains a general GFP expressing vector for mammalian cells (pCMV-EGFP or pCAGGS-EGFP). In this case, please use it for each of the parameters. The amount that should be used is in the protocol sheet, typically 10 μg in 100 μL of cells at 1 x 10⁶ concentration.
6. Avoid bubbles when loading into the cuvette.
7. Do not lower the DNA amount until pulsing parameters have been fully optimized.
8. It is best to dry down the DNA pellet and resuspend in ddH2O to avoid any molecules that may affect the ionic conditions (such as EDTA).
9. Immediately plate cells after electroporation and before pulsing the next cuvette. Don’t allow cells to remain in the cuvettes any longer than necessary following electrical pulsing.
10. **Imperative** It’s important to properly resuspend cells within the cuvette to eliminate clumping that can occur both before and after electric pulsing. This clumping can drastically reduce cell viability and/or transfection efficiency, and often leads inconsistent data. Please use a gel-loading pipette tip to load and retrieve all cells from the cuvette. Repeated, gentle pipetting through the small-bore tip will help break up clumps.

More Questions? Please ask us

Adherent Cell Electrode Method

The NEPA21 can accept hundreds of electrode accessories for dozens of applications. The family of adherent cell electrodes provide a means to use electro-kinetic transfer on cells that are still in an adherent state. We have an in-house written protocol available to any lab that owns a NEPA21 system. Please ask us for this document.

The NEPA21 adherent electrode protocols usually use 3- or 4-phases of electrical pulsing, including a Poring Pulse Phase to “open holes” in the membrane, followed by a Transfer Pulse Phase to move the charged molecules (usually DNA, RNA or proteins) into the cell. Both phases are essential for high cell viability and transfection efficiency.

Advantages:

• No requirement for special buffers or expensive reagents
• Fully adjustable electrical pulsing parameters
• Non-capacitor design for handling physiological salt conditions
• Can be used to transfect large plasmids (10+ kb)
• Works with proteins, RNA and gene editing complexes
• Proven on many cell lines and cell types.

Key publications: The NEPA21 has been referenced in more than 800 publications since 2012. We would be happy to help you find the publications most pertinent to your lab’s particular needs.

Using the NEPA21 for adherent electrode method:

Ask us for both a written protocol and also our suggested pulsing parameters for your lab’s particular cells of interest. These can be supplied to any NEPA21 lab.

Cell Types:

Hundreds of cell lines and cell types have been demonstrated to work with the NEPA21 system. Below is a quick summary; click here for an up-to-date list of dozens of cell types with images and expected results.

• Primary neuronal cells
• Primary epithelial cells
• Primary endothelial cells
• Primary stem cells

Electrode Types:

CUY900-13-3-5 Electrode for 24-well culture plate

• 300 μL volume
• buffer type: Opti-MEM
• cells should be in a healthy logarithmic growth phase
• 150 μg of plasmid DNA
• target Impedance reading is 170 Ω to 350 Ω

CUY900-5-2-3 Electrode for 96-well culture plate

CUY900-20-5-3 Electrode for 12-well culture plate

CUY900-32-5-5 Electrode for 6-well culture plate

Tips for Success:

1. Please ask us for a spreadsheet with recommended, field-tested, parameter sets to try. When first optimizing a new cell line or cell type, use ALL of the recommended pulsing parameters for that cell type.
2. Always record output electrical data – especially the IMPEDANCE value. We are happy to offer suggestions for further optimization, but we can only help you interpret your results when the electrical data is available.
3. We recommend performing the transfection in serum-free (or at minimum serum-reduced) Opti-MEM. The cells should be washed at least twice to ensure good electric-field formation.
4. Use a high quality source for reagents. In the case of DNA plasmids purify using an endotoxin-free kit (see our suggested kit). For gene editing reagents we recommend using the Cas9 protein instead of mRNA.
5. If you are using your own plasmid vector, please ensure that it has already been fully qualified and works for other transfection methods. Otherwise, ask us about our control DNA, which contains a general GFP expressing vector for mammalian cells (pCMV-EGFP or pCAGGS-EGFP). In this case, please use it for each of the parameters. The amount that should be used is in the protocol sheet, typically 150 μg in 300 μL in one well of a 24-well plate.
6. Avoid bubbles when loading into the wells.
7. Do not lower the DNA amount until pulsing parameters are fully optimized. You can reuse the same DNA-EP buffer on up to 12 wells if desired.
8. It is best to dry down the DNA pellet and resuspend in ddH2O to avoid any molecules that may affect the ionic conditions (such as EDTA).
9. Use the recommended sterilization procedure for the electrode as listed in the protocol. Or see our recommendations here.

More Questions? Please ask us

“Technique for Animal Knockout system by Electroporation” or TAKE Method

Developed in 2014 by Takehito Kaneko at Kyoto University in Japan, this technique replaces microinjection for the production of transgenic animals. This method is simpler, faster, and more efficient for the introduction of gene editing reagents (e.g. CRIPSR-Cas9) into mouse, rat, and other animal zygotes.

Advantages:

• No requirement for microinjection
• No requirement to remove or weaken the zona pellucida
• No special training required
• Process more than 100 embryos in 5 minutes as compared with microinjection

Key publications:

Simple Knockout by Electroporation of Engineered Endonucleases into Intact Rat Embryos (2014)

Simple Genome Editing of Rodent Intact Embryos by Electroporation (2015)

Genome Editing in Mouse and Rat by Electroporation (2017)

Using the NEPA21 for TAKE method:

Ask us for our “in house” protocol and pulsing parameters. These can be supplied to any NEPA21 lab.

Animal Types:

The TAKE method has been demonstrated successfully in the following animal models:

• Mouse
• Rat
• Cat
• Pig

Electrode Types:

CUY505P5

• 5 mm gap high capacity electrode
• process between 20 and 100 embryos at a time
• requires 45 μL of reagent (1.2 μM Cas9 protein, 6 μM crRAN:tracrRNA, 50 ng/μL lssDNA or 300 ng/μL ssODN)
• target Impedance reading is 480 Ω to 520 Ω

CUY501P1-1.5

• 1 mm gap low capacity electrode
• process between 5 and 50 embryos at a time
• requires 5 μL of reagent (1.2 μM Cas9 protein, 6 μM crRAN:tracrRNA, 50 ng/μL lssDNA or 300 ng/μL ssODN)
• target Impedance reading is 180 Ω to 220 Ω

Tips for Success:

1. Always record output electrical data – especially IMPEDANCE!
2. Use a high quality source for Cas9 protein instead of mRNA
3. Knock-in/out size should be below 500 nt for the initial application development in your lab. Larger inserts are possible, but only up to 1.5 kb. It’s best to develop good skills for this technique first.
4. This protocol is NOT compatible with plasmid DNA or with dsDNA.
5. Work quickly with the 1mm gap zygote electrode. We’ve had feedback that evaporation can cause the impedance to rise. You may need to add back reagent after processing a batch or two of zygotes. This is less of a problem with the 5 mm gap CUY505P5 electrode.
6. When loading the zygotes, make sure to: i) keep them as centered as possible to prevent them from touching the electrode plates (), ii) never let them touch each other or pile up, iii) spread them as evenly as possible up and down the center of the bath, and iv) prevent surface tension-induced beading by spreading reagent evenly and thoroughly between electrode plates.
7. Use fresh, healthy zygotes. Examine zygotes about 10 minutes after pulsing. Discard zygotes that start to lose sharp edges or begin to flatten; these will likely die.
8. The RNP complex solution can be reused for processing larger numbers of embryos.

More Questions? Please ask us

“Genome-editing via Oviductal Nucleic Acids Delivery” or GONAD Method

Developed in 2015 by Masato Ohtsuka (Tokai University School of Medicine) and Masahiro Sato (Kagoshima University) as an in vivo-only electroporation technique to replace microinjection in the production of transgenic animals. As compared with the TAKE method, GONAD is simpler and faster for the introduction of gene editing reagents (e.g. CRIPSR-Cas9) into mouse, rat and other animal zygotes.

Advantages:

• No requirement for microinjection
• No need to isolate and transfer embryos
• No requirement to prepare vasectomized male mice and pseudopregnant recipient mice
• Process each female mouse in 10 minutes
• Helps meet the 3R principle; Reduction, Replacement and Refinement

Key publications:

GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice (2014)

Successful production of genome-edited rats by the rGONAD method (2018)

i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats (2018)

Using the NEPA21 for GONAD method:

Ask us for our “in house” protocol and pulsing parameters. These can be supplied to any NEPA21 lab.

Animal Types:

The GONAD method has been demonstrated successfully in the following animal models:

• Mouse
• Rat

Electrode Types:

CUY652P2.5X4

• concave “cupped” platinum electrode specially designed for this technique
• target tissue is ampulla
• process both uterine horns of a ovulating female in under 10 min.
• requires 1.5 μL of reagent per uterine horn (1.2 μM Cas9 protein, 6 μM crRAN:tracrRNA, 50 ng/μL lssDNA or 300 ng/μL ssODN)
• target Impedance reading is 90 Ω to 250 Ω

Tips for Success:

1. Always record output electrical data–especially IMPEDANCE.
2. Use a high quality source for Cas9 protein instead of mRNA
3. Knock-in/out size should be below 500 nt for the initial application development in your lab. Larger inserts are possible, but only up to 1.5 kb. It’s best to develop good skills for this technique first.
4. This protocol is NOT compatible with plasmid DNA or with dsDNA.
5. The CUY652 tweezer electrodes often need to be squeezed until the opposing electrodes nearly touch. Quickly check impedance to confirm correct range and then immediately use the foot pedal to pulse.
6. Always choose the correct timing so that the cumulous cells are well separated from the zygotes. This is typically at 0.7 days (4:00 pm).
7. Please review the videos for GONAD technique.Check out these two short videos for performing rGONAD (this is also applicable in mice), and our in-house video supplied by Hua Yue Enterprise Holdings, Ltd. (Nepa Gene distributor for China).

More Questions? Please ask us