The TAKE method [13 (link)] was used to introduce Cas9 mRNA, gRNA and ssODN into intact mouse and rat embryos. This method introduced endonucleases into intact embryos using a super electroporator NEPA 21 (NEPA GENE Co. Ltd., Chiba, Japan). Briefly, pronuclear-stage embryos were placed in a line on the glass chamber between 5mm gap platinum metal plates (CUY520P5, NEPA GENE Co. Ltd.) that were filled with 100 μL PBS containing Cas9 mRNA, gRNA and ssODN at various concentrations. The poring pulse was set to voltage: 225 V, pulse width: 2.5 ms, pulse interval: 50 ms, and number of pulses: +4. The first and second transfer pulse were set to voltage: 20 V, pulse width: 50 ms, pulse interval: 50 ms, and number of pulses: ±5. After electroporation, all embryos were cultured before embryo transfer.
Cuy520p5
Manufactured by Nepa Gene
Sourced in Japan
The CUY520P5 is a laboratory equipment designed for DNA extraction and purification. It is a compact and automated system that utilizes a proprietary filtration method to isolate high-quality DNA from a variety of sample types. The core function of the CUY520P5 is to provide a reliable and efficient DNA extraction solution for research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Nepa21, by Nepa Gene (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Nuclepore track etch membrane, by Cytiva (1 mentions)
Nuclease free water, by Thermo Fisher Scientific (1 mentions)
F344 jcl, by CLEA Japan (1 mentions)
Cuy505p5, by Nepa Gene (1 mentions)
Nepa21 super electroporator, by Nepa Gene (1 mentions)
4 protocols using cuy520p5
1
Efficient Genetic Modification of Embryos
2
Retinal Electroporation and Ex Vivo Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Retinas were dissected in DMEM/F12 1:1 buffer (Gibco™) from newborn (P0) WT and Crx mutant mice. For each replicate, three retinas were transferred into the electroporation chamber (CUY520P5, NEPA GENE Co. Ltd) filled with the prepared MPRA plasmid DNA solution. The retinas were electroporated with the Electro Square Porator™ (ECM®830, BTX) with settings: LV, V: 30 Volts, Pulse Length: 50 msec, # Pulses: 5, Interval: 950 msec. The electroporated retinas were washed once in the retinal ex plant culture medium, carefully transferred onto a 0.2μm 25mm Nuclepore™ Track-Etch Membrane (Whatman®) and cultured for 8 days in the incubator (37°C, 5% CO2) with the retinal ex plant culture medium (DMEM/F12 1:1, 10% HI-FBS, 1xPenicillin-Streptomycin). The day-8 ex plant retinas were collected, washed once in cold PBS, and stored in 30μl Nuclease-Free Water (Invitrogen™) at −80°C before ready for TRIzol extraction.
3
CRISPR Delivery to Mouse Zygotes via TAKE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TAKE method10 (link) was applied to deliver CRISPR components to zygotes of mice ex vivo using an electroporator NEPA21 (NEPA GENE, Chiba, Japan). Briefly, pronuclear-stage embryos were transferred into the 5 mm gap-electrode (CUY520P5, NEPA GENE) filled with 50 µL CRISPR reagent containing Cas9 protein, gRNA, and AAV donor. The poring and transfer pulse were set as previously described by Kaneko et al.10 (link). After electroporation, zygotes were cultured to be embryos in the 2-cell stage.
4
Efficient CRISPR-Cas9 Genome Editing in Rat Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
F344/Jcl (CLEA Japan, Inc. Tokyo, Japan) rat embryos were collected from 7 to 8 weeks of age females that were superovulated by administration of 150 U/kg of PMSG followed by 75 U/kg of HCG. After natural mating, pronuclear-stage embryos were collected from oviducts of the females and cultured in a modified Krebs–Ringer bicarbonate medium (ARK Resource, Kumamoto, Japan). For electroporation, 50–100 embryos 3–4 h after collecting were placed into the chamber with 40 µl of serum free media (Opti-MEM Thermo Fisher Scientific MA, USA) containing 400 ng/µl Cas9 mRNA, 200 ng/µl gRNA. They were electroporated with a 5 mm gap electrode (CUY505P5 or CUY520P5 Nepa Gene, Chiba, Japan) in a NEPA21 Super Electroporator (Nepa Gene, Chiba, Japan). The poring pulses for the electroporation were voltage 225 V, pulse width 2.0 ms for rat embryos, pulse interval 50 ms, and number of pulses + 4. The first and second transfer pulses were voltage 20 V, pulse width 50 ms, pulse interval 50 ms, and number of pulses + 5. Embryos that developed to the two-cell stage after the introduction of RNAs were transferred into the oviducts of female surrogates anesthetized with isoflurane (DS Pharma Animal Health Co., Ltd., Osaka, Japan).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!