Adh5−/− and Aldh2+/KI mice have been described previously in ref. 11 (link). To generate Csb-deficient mice, the following reagents were purchased: Cas9 Nuclease V3, tracrRNA and crRNA (Integrated DNA Technologies). Pronuclear-stage mouse embryos were prepared by thawing frozen embryos (CLEA Japan), then culturing them in potassium simplex optimized medium (KSOM, ARK Resource). For electroporation, 100–150 embryos (1 h after thawing) were placed into a chamber with 40 μl of serum-free medium (Opti-MEM, Thermo Fisher Scientific) containing 100 ng μl−1 Cas9 Nuclease V3 and 100 ng μl−1Csb gRNA. They were then electroporated with a 5-mm gap electrode (CUY505P5, NepaGene) in a NEPA21 Super Electroporator (NepaGene). The pulses for the electroporation had a voltage of 225 V, pulse width of 1 ms for mouse embryos, pulse interval of 50 ms, and the numbers of pulses was 4. The first and second transfer pulses had a voltage of 20 V, pulse width of 50 ms, pulse interval of 50 ms, and the number of pulses was 5. Mouse embryos that developed to the two-cell stage after electroporation were transferred into the oviducts of female surrogates anaesthetized with sevoflurane or isoflurane (Mylan). gRNA sequence information is listed in Supplementary Table 7 .
Cuy505p5
Manufactured by Nepa Gene
Sourced in Japan
The CUY505P5 is a laboratory equipment designed for scientific applications. It serves as a power supply unit, providing a stable and reliable source of electrical power for various laboratory instruments and experiments. The core function of the CUY505P5 is to convert alternating current (AC) to direct current (DC) and regulate the voltage and current output to meet the specific requirements of the connected equipment.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (5 mentions)
Nepa21 super electroporator, by Nepa Gene (3 mentions)
Crrna, by Integrated DNA Technologies (1 mentions)
Cas9 nuclease v3, by Integrated DNA Technologies (1 mentions)
Tracrrna, by Integrated DNA Technologies (1 mentions)
Nepa21, by Nepa Gene (1 mentions)
Opti mem, by Nepa Gene (1 mentions)
F344 jcl, by CLEA Japan (1 mentions)
Cuy520p5, by Nepa Gene (1 mentions)
Mmn 1, by Narishige (1 mentions)
Mhw103, by Narishige (1 mentions)
Im 31, by Narishige (1 mentions)
Pc 100, by Narishige (1 mentions)
5 protocols using cuy505p5
1
CRISPR-Mediated Generation of Csb-Deficient Mice
2
CRISPR Electroporation of Zygotes
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Fertilized eggs were suspended in Opti-MEM (Gibco, 31985-070) in the presence of RNP and placed on microscope slides with platinum-plated electrodes (NEPA GENE, CUY505P5). RNP was delivered into zygotes using a NEPA 21 Super Electroporator (NEPA GENE) according to the recommended protocol. Four days after incubation of the eggs in KSOM medium at 37 °C, cells at the morula stage were harvested for preparation of genomic DNA.
3
Improved Zygote Electroporation Protocol
4
Efficient CRISPR-Cas9 Genome Editing in Rat Embryos
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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).
5
Microinjection and Electroporation of Tardigrades
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Glass capillaries (GD-1, NARISHIGE) were pulled by a puller (PC-100, NARISHIGE); the temperatures were set at 66.2 and 62.0 °C. Our microinjection system consists of an inverted microscope (AXIO Vert. A1, Zeiss) equipped with an injector (IM-31, NARISHIGE) and manipulators (MMN-1 and MHW-103, NARISHIGE). Tardigrades were mounted as described previously by Tenlen et al. for RNAi experiments without anesthesia (Fig. 1B ) (54 (link), 55 (link)). After the microinjections, individuals were collected and transferred to a cuvette (CUY505P5, NEPA GENE) for electroporation using a super electroporator (NEPA21 type 2, NEPA GENE). The poring pulse was emitted twice at 250 V for 5 ms with a 50-ms interval, and the transfer pulse was emitted five times at 30 V for 50 ms with a 50-ms interval. Tardigrades were maintained with algal food on an agar plate until observation at 17 °C.
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