Free NEPA21 Demo and Trial :-Zygote Electroporation for Transgenic Animal Production
Transgenic Animal Production / Zygote Electroporation
Compared to other devices on the market, the NEPA21 system offers the researcher a level of previously unavailable control over energy delivery to the electroporation target. This control is generated via unique electroporation pulse-output configurations, client-confirmed protocols and application-customised electrodes.
With this market-leading control and (user-independent) reproducibility of the technique, it is now possible to apply electroporation techniques to applications previously considered too sensitive for electroporation methodologies. One such application is Zygote Electroporation for Transgenic Animal Production
NEPA21 TRIAL / DEMONSTRATION Offer
Our confidence in our NEPA21 system is such that we offer a FREE ‘try-before-you-purchase’ DEMONSTRATION and TRIAL offer.
If you would like to avail of this offer, please click on this link and Request a NEPA21 TRIAL.
Trial terms and conditions are straightforward. Please click here for further details: NEPA21 TRIAL Terms and Conditions
Please also email sales@sonidel.com if you would like to receive copies of our most recent Genome Editing publications:
– NEPA21 Publication List – cell cultures – 1) Stem Cells
– NEPA21 Publication list – cell cultures – 2) Organoids
– NEPA21 Publication List – cell cultures – 3 Human
– NEPA21 Publication List – cell cultures – 4) Mouse
– NEPA21 In Vivo & Ex Vivo Electroporation
– The latest SONIDEL Cell Transfection Database (Detailing Primary Cells and Cell Lines optimised with the NEPA21 system)
– A third-party (IDT) comparison of the NEPA21 against competing devices (GenePulser and Amaxa 4D) for Genome Editing Efficiency
Zygote Electroporation for Transgenic Animal Production
With the NEPA21, it is possible to electroporate Zygotes:
– Ex Vivo and
– In Vivo
Choice of methodology is dictated by the level of micro-manipulation skill accessible in the laboratory and access to animal license certification.
If a laboratory has minimal previous experience of Zygote Electroporation, we recommend the Ex Vivo (TAKE) Method/CLICK Method.
Alternatively, for a laboratory with access to relevant animal licenses and the required manipulation skills, we recommend the In Vivo (iGONAD) Method.
Traditionally, researchers have used microinjection techniques for transgenic animal production. While a robustly successful technique, it has intrinsic disadvantages. The NEPA21 electroporation methodologies improve upon microinjection as follows:
– No requirement for microinjection
– No requirement to remove the Zona Pellucida
– No need to weaken the zona pellucida by pre-treatment with Tyrode solution
– No need for specialist training – all team members can perform the technique
– The Ex Vivo TAKE Method only takes 5 minutes to electroporate up to 150 embryos
– With the In Vivo iGONAD Method, embryos can be electroporated in-situ in the Oviduct, obviating the need for the ex vivo handling-steps and stages of the Ex Vivo TAKE Method
– For KNOCK-OUT and KNOCK-IN applications, results are more reproducible and, in many cases, better than with microinjection
Note this link to the Charles River blog page promoting our NEPA21 system as the best in the field.
Note also, further 3rd party links to full NEPA21 protocols, this time, on the IDT website:
“Here you can access protocols for Mouse Zygote Electroporation and Human-induced iPS Cells using the NEPA21 Electroporator”.
(The ‘Human-induced iPS Cells’ link is a step-by-step protocol elucidation).
Application-Customised Electrode Options
To further enhance the efficiency of the NEPA21 system for Zygote Electroporation, we have developed application-specific electrodes.
For the Ex Vivo (TAKE) Method, we offer two different electrode size options:
– the CUY501P1-1.5 electrode is 1mm in gap width and is a small volume solution. It accommodates 5ul of CRISPR solution in its chamber and electroporates from 5-50 embryos at a time.
– the CUY505P5 electrode is 5mm in gap width and is a larger volume solution. It accommodated 45ul of CRISPR solution in its chamber and electroporates form 20-150 embryos at a time.
For the In Vivo (iGONAD) Method, we offer two different electrode size options:
The CUY652P2.5×4 is concave in shape and optimised to fit a MOUSE oviduct.
The CUY652P3x4.5 is concave in shape and optimised to fit a RAT oviduct
Click Method
For generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA–2ssODN)
The Click Method. It is an evolution of the TAKE Method
In terms of the available art, we reference the very well know article:
Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology
(doi: https://doi.org/10.1101/393231, (the Node 03 Oct., 2018):
The article cites a previous article that reports up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA–2ssODN).
The article re-evaluates the reported method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harboured 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method.
The reported analysis indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.
For your information, we also reference another article published by a NEPA21 client, Prof. Tomoji Mashimo:
Click: one-step generation of conditional knockout mice
This article advanced the art even further and demonstrates successful conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA–2ssODN).
Pro. Mashimo performs HR with RNP and short oligo using the NEPA21 and obtain high efficiency.
He confirms that his lab is also successfully performing HR with long single stranded DNA even for the Rosa locus.
He can KNOCK-IN less than 1.2 kb cassette with the NEPA21
Ex Vivo TAKE and CLICK Methods
These methodologies represent the most widely used methodology to create genome-edited animals. They employ 3 major ex vivo embryo handling steps:
1. Isolation of the zygotes from a pregnant female (previously mated with a male)
2. Zygote microinjection of genome editing components and
3. Surgical transfer of microinjected zygotes into the oviduct of a pseudopregnant female
Each step requires a high level of technical expertise and proficiency, expensive apparatus (micromanipulator) and relevant animal licences.
In Vivo iGONAD Method
The improved i-GONAD (MOUSE) and r-GONAD (RAT) techniques are a step-by-step methodology for a novel and improved in vivo genome editing system that does not require ex vivo handling of embryos.
i-GONAD (improved GONAD) for Mice and r-GONAD for Rats
⦁ The methodology is called GONAD (Genome-editing via Oviductal Nucleic Acids Delivery) and details the techniques:
⦁ i-GONAD (improved GONAD) for MICE, and
⦁ r-GONAD for RATS
The very first GONAD applications (Ohtsuka: using Cas9 mRNA and sgRNA) report genome editing efficiency of approximately 25%.
⦁ The new improved i-GONAD and r-GONAD application (Matsuyama: using Cas9 protein and crRNA/tracrRNA complex) report:
⦁ Knock-out: 50-100% and
⦁ Knock-in: 15-40%
It is particularly applicable for laboratories:
⦁ where traditional gene targeting using ES Cells is not well established in mammals such as guinea pig, hamster, cow, pig and other animals
⦁ keen to minimise their 3R footprint – as iGONAD does not require the euthanasia of pregnant females unlike in traditional methodologies where females need to be sacrificed for isolating zygotes (for introduction of genome editing components ex vivo). In addition, some females who undergo iGONAD can deliver the pups.
i-GONAD Video Support
On the following link to our website, one can access an Aide-memoire for i-GONAD Electroporation. This document was prepared after we delivered Zygote Workshops at two client sites in Lisbon and Barcelona. The aide-memoire is a step-by-step demonstration of the technique by Dr. Matsuyama and his team and is augmented with:
⦁ detailed background and supporting notes on the critical steps to ensure success and
⦁ videos of critical steps execution (filmed at the Workshops)
In addition, NEPA21 clients can access via the following link an even more comprehensive and detailed High-Definition Video of the i-GONAD Protocol and Technique. Access can be obtained by requesting one-time-password access from sales@sonidel.com.
SONIDEL can deliver workshops at client laboratory/institution for both the TAKE and i-GONAD Methods. With respect to both methods, we can provide step-by-step protocols and high-definition video resource for each application-step of the protocols.
Recent Publications
1. Dumeau et al. 2019
⦁ Title: Introducing gene deletions by mouse zygote electroporation of Cas12a/Cpf1
⦁ Materials: Cas12a RNP
⦁ Targets: Mouse zygotes
⦁ Electrode: CUY501P1-1.5 (small volume electrode option)
⦁ EP conditions: See Table 1 of publication:
⦁ Summary:
⦁ Poring Pulse: 30V / 3ms Length / 100ms Interval / 6 Pulses / Decay Rate 10% / Polarity Switch +
⦁ Transfer Pulse: 5V / 50ms Length / 50ms Interval / 5 Pulses / Decay Rate 40% / Polarity Switch =/-
Notes:
⦁ The article compares zygotes with/without an intact zona pellucida, and there were no significant differences
⦁ The paper states: “The NEPA electroporation system enables measurements of sample conductance and a precise reading of electrical intensity and energy delivered, which improves the reproducibility of the experimental conditions.”
2. Kim et al. 2019
⦁ Title: Electroporation of AsCpf1/RNP at the Zygote Stage is an Efficient Genome Editing Method to Generate Knock-Out Mice Deficient in Leukemia Inhibitory Factor
⦁ Materials: AsCpf1 (Cas12a)/RNP
⦁ Targets: Mouse zygotes
⦁ Electrode: CUY520P5 (large volume electrode option)
⦁ EP conditions: Not available from the publication
Notes:
⦁ The results were compared between AsCpf1/mRNA and AsCpf1/RNP. See Table 1 of publication
⦁ The paper states: ”we established an easy, fast, and technically less demanding method to produce KO mice using electroporation of the Cfp1/RNP system. Electroporation of AsCpf1/RNP at the zygote stage is an efficient genome editing method to produce KO mice.”
Click for NEPA21 Publications – Zygote Electroporation
Click for Full NEPA21 List of publications NEPA21 Cell Transfection Database
Further information:
- NEPA21 List of publications , it lists relevant ex vivo Zygote publications (for Mouse/Rat/Marmosets/Pigs and Monkeys and in vivo Zygote (in the Oviduct – iGONAD Method) publications (for Mouse/ Rat and Hamster)
- Review of NEPA21 – a third-party (IDT) comparison of the NEPA21 against competing devices (GenePulser and Amaxa 4D) for Genome Editing Efficiency
- Kaneko – Genome Editing in Mouse and Rat by Electroporation – Chapter 7
- NEPAGENE zygotes genome editing methods