Rat-Knockout-Zygote_and_Oocyte_Electroporation

Rat-Knockout: Zygote and Oocyte Electroporation

Please note this link to the latest Takehito Kaneko publication: Genome Editing in Mouse and Rat by Electroporation
The publication appears as Chapter 7 in the Izuho Hatada edited:
Genome Editing in Animals: Methods and Protocols
Methods in Molecular Biology, vol. 1630,
DOI 10.1007/978-1-4939-7128-2_7, © Springer Science+Business Media LLC 2017

The chapter presents in step-by-step detail (for both Mouse and Rat) a new technique called ‘Technique for Animal Knockout system by Electroporation’ (TAKE Method), which produces genome-edited rodents by direct introduction of engineered endonucleases into intact embryos using electroporation.

Knock-out/knock-in mice and rats can be produced by introducing engineered endonucleases into intact embryos using this TAKE electroporation system.

There is absolutely NO requirement for microinjection
There is absolutely NO requirement to remove the Zona Pellucida
There is absolutely NO need to weaken the zona by pre-treatment with Thyrode solution
Any member of your team can now perform Zygote EP – there is NO need for specialist training
It only takes 5 minutes to electroporate up to 100 embryos at a time.

Video of Takehito Kaneko performing the TAKE Method:
TAKE Method – Video 1
TAKE Method – Video 2
TAKE Method – Video 3
TAKE Method – Video 4
TAKE Method – Video 5

TAKE Method Pointers
–  Dr Kaneko points out that immediately after EP, he cleans up.  That process takes 10 minutes.
–  He then examines the embryos again.  The purpose of waiting 10 minutes to check the embryos is that if they have been damaged during EP, then within 10 minutes post EP they will be dead.
–  As a rule of thumb: if the embryo is OK 10 minutes after EP, then, invariably it is fine the next day.
–  After EP, healthy embryos have a shadow around their edges. Dead embryos are flatter and more spread out.
–  After inspection. healthy embryos are transferred to culture media and then cultured over-night and transferred to the animals the next day (when they have developed to 2 cells).
–  Dr Kaneko recommends the use of Fresh embryos (as the success rate with frozen embryos is lower than with fresh embryos).
–  It is possible that one could further optimise the EP conditions (using the NEPA21) and perhaps improve the frozen embryo results.  But using this TAKE protocol, it is recommended that fresh embryos are used.
–  When placing the embryos in the electrode, try to place them in the middle of the electrode.  As EP produces bubbles, if the embryos are placed too close the electrode, the embryos disappear (and are vaporised) with the bubbles that come off the electrode blades during electroporation.
–  To clean the CUY520P5 electrode.  Simply use tissue paper.
–  If the electrode is particularly dirty, clean with a 70% ethanol solution.

Here is a summary of what we recommend:
CUY5001P1-1.5 electrode
Media and Solutions
– Materials: CRISPR Cas9 mRNA, gRNA
– Solution in the chamber: 5 ul (Cas9 mRNA: 400ng/ul, gRNA: 200ng/ul)
– No of Zygotes: 5-40 embryos each tested condition

Kaneko uses our CUY520P5 5mm gap electrode:
CUY520P5 electrode
Media and Solutions
– Materials: CRISPR Cas9 mRNA, gRNA
– Solution in the chamber: 40 ul (Cas9 mRNA: 400ng/ul, gRNA: 600ng/ul)
– No of Zygotes: 30-100 embryos tested each condition

40ul is recommended for the CUY520P5 and CUY501P1-1.5 larger electrode configurations.
5ul is recommended for the CUY5001P1-1.5 and CUY501P1-1.5 smaller electrode configurations.

Other Relevant Publications citing the NEPA21 and our electrodes
Please note the Nature Biotechnology publication from Prof. Kyoungmi Kim:
High efficient RNA-guided base editing in mouse embryos
Nature Biotechnology 35, 435–437 (2017) doi:10.1038/nbt.3816, 27 February 2017

“Base editors (BEs) composed of a cytidine deaminase fused to CRISPR–Cas9 convert cytidine to uridine, leading to single-base-pair substitutions in eukaryotic cells. We delivered BE mRNA or ribonucleoproteins targeting the Dmd or Tyr gene via electroporation or microinjection into mouse zygotes. F0 mice showed nonsense mutations with an efficiency of 44–57% and allelic frequencies of up to 100%, demonstrating an efficient method to generate mice with targeted point mutations”.

Please also note that another client reports he has successfully electroporated a large piece of DNA (cre-loxp system).

Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoprotein
6 June 2016
Jin-Soo Kim, Center for Genome Engineering, Institute for Basic Science, Seoul, Republic of Korea.

This paper demonstrates the use of the NEPA21 system for mouse zygote electroporation of Cas9 protein (not mRNA).
The author investigated whether both Cas9 RNPs and Cpf1 RNPs could be delivered into animal embryos by electroporation rather than microinjection.
The author points out that while microinjection is widely used for making transgenic or gene knockout animals, microinjecting single-cell embryos individually is technically difficult, requiring extensive training, and is labour-intensive.

“A  skilled researcher can handle hundreds of embryos per day at best.  Using the [NEPA21] electroporation method, we simultaneously delivered RNP to multiple embryos, up to 50 at once, within 5 minutes.”

The researcher electroporated SpCas9 RNPs targeting the Vegfa or Foxn1 locus in the mouse genome into 50 embryos for each locus in a chamber [CUY520P5] electrode, which were then cultured to blastocysts.

Results
“12 of 15 (80%) or 11 of 11 (100%) blastocysts obtained had mutations at the Vegfa or Foxn1 target site, respectively… suggesting that electroporation  is an  efficient method of delivering Cas9 RNPs into animal embryos.”

Summary
“… our results show that electroporation [using the NEPA21] of AsCpf1 RNPs resulted in efficient and specific genome editing in mouse embryos.  RNPs are as effective as mRNA or plasmids, but are degraded rapidly by endogenous proteases and RNases in cells, and have been previously shown to reduce off-target effects and mosaicism.  Unlike microinjection, electroporation [with the NEPA21] is easy to carry out, fast, and scalable.  Up to 50 embryos can be electroporated simultaneously.  We propose that electroporation of Cpf1 RNPs is a potentially useful new method for genome editing in animals.”

Please note that this client previously used the Nucleofector system (prior to purchasing the NEPA21 system).
This is why, in this paper, the author cites the Nucleofector device for cell culture, NIH/3T3.
He now uses his NEPA21 system for cell culture work for NIH/3T3.
With the NEPA21 system we achieve for NIH/3T3: 100% Viability and 90% Transfection Efficiency

TAKE METHOD: Technique for animal knockout system by electroporation
The methodology outlined initially in Simple knockout by electroporation of engineered endonucleases into intact rat embryos (2014) was further enhanced and formalised as the TAKE Method in Dr kaneko’s subsequent November 2015 article:
Simple Genome Editing of Rodent Intact Embryos by Electroporation
10 Nov 2015
Takehito Kaneko1, Tomoji Mashimo2
1 Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, 606–8501, Japan
2 Institute of Experimental Animal Sciences, Faculty of Medicine, Osaka University, Osaka, 565–0871,
Japan

Electrode: Our CUY520P5
Materials: CRISPR Cas9 mRNA, gRNA
Solution concentration in the chamber of the CUY520P5: 40 ul: [16 ug mRNA in 20 ul Opti-MEM] + [24 ug gRNA in 20 ul Opti-MEM]
*The RNA solution can be reused during the test.
Target:                              Zygotes without weakening the zona pellucida
Number of Embryos:     30-50 embryos each electroporation

Simple knockout by electroporation of engineered endonucleases into intact rat embryos
Takehito Kaneko, Tetsushi Sakuma, Takashi Yamamoto & Tomoji Mashimo
Scientific Reports 4, Article number: 6382 (2014)
doi:10.1038/srep06382
Further Information:
– 30-50 embryos are used at one EP.
– the 100ul mRNA solution  in the CUY520P5 can be reused.
– for Cas9 mRNA: “200 ug mRNA in 50 ul PBS (-)” + “100 ug gRNA in 50 ul PBS (-)”.
– Electroporation with mRNA have been done successfully, but electroporation with Plasmid DNA has not yet been successful

Please contact us for a demonstration: sales@sonidel.com