Standard bipolar in utero electroporation of the somatosensory cortex was performed as previously described (Bony et al., 2013 (link); Szczurkowska et al., 2016 (link)). Timed-pregnant Sprague Dawley rats (Harlan Italy SRL, Correzzana, Italy) were anaesthetized at E17.5 with isoflurane (induction, 3.5%; surgery, 2.5%), and uterine horns were exposed by laparotomy. Expression vectors (1–2 μg μl−1/Vector in water) and dye Fast Green (0.3 mg ml−1; Sigma, St. Louis, MO) were injected (5–6 μl) through the uterine wall into one of the embryo’s lateral ventricle by a 30-G needle (Pic indolor, Grandate, Italy). Each embryo’s head was held between tweezer-type electrodes (10 mm diameter; Nepa Gene, Chiba, Japan) across the uterus and five electrical pulses (amplitude, 50 V; duration, 50 ms; intervals, 100 ms) were delivered with a square-wave electroporation generator (CUY21EDIT; Nepa Gene). Uterine horns were returned into the abdominal cavity, and embryos continued their normal development.
Cuy21edit
Manufactured by Nepa Gene
Sourced in Japan
The CUY21EDIT is a versatile lab equipment designed for genetic engineering applications. It features a compact and ergonomic design, allowing for efficient and precise genome editing. The core function of the CUY21EDIT is to facilitate the introduction of targeted modifications within DNA sequences, enabling researchers to study and manipulate genetic information.
Automatically generated - may contain errors
Lab products found in correlation
Fast green, by Merck Group (6 mentions)
Cuy650p5, by Nepa Gene (3 mentions)
30 g needle, by Merck Group (2 mentions)
Tweezer type electrodes, by Nepa Gene (2 mentions)
Pcag ires egfp, by Merck Group (2 mentions)
Standard forceps type circular electrodes, by Nepa Gene (2 mentions)
Cuy650p3, by Nepa Gene (2 mentions)
Negative pole disc type electrodes, by Nepa Gene (2 mentions)
C57bl 6 mice, by Charles River Laboratories (2 mentions)
Endofree plasmid kit, by Qiagen (1 mentions)
Im 30, by Narishige (1 mentions)
Glass capillaries, by Harvard Apparatus (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Vibratome vt1000, by Leica (1 mentions)
Cuy650 5, by Nepa Gene (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
Auto clip, by Fine Science Tools (1 mentions)
24 protocols using cuy21edit
1
In Utero Electroporation of Somatosensory Cortex
2
In Utero Electroporation of Somatosensory Cortex
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Standard bipolar in utero electroporation of the somatosensory cortex was performed as previously described (Bony et al., 2013 (link); Szczurkowska et al., 2016 (link)). Timed-pregnant Sprague Dawley rats (Harlan Italy SRL, Correzzana, Italy) were anaesthetized at E17.5 with isoflurane (induction, 3.5%; surgery, 2.5%), and uterine horns were exposed by laparotomy. Expression vectors (1–2 μg μl−1/Vector in water) and dye Fast Green (0.3 mg ml−1; Sigma, St. Louis, MO) were injected (5–6 μl) through the uterine wall into one of the embryo’s lateral ventricle by a 30-G needle (Pic indolor, Grandate, Italy). Each embryo’s head was held between tweezer-type electrodes (10 mm diameter; Nepa Gene, Chiba, Japan) across the uterus and five electrical pulses (amplitude, 50 V; duration, 50 ms; intervals, 100 ms) were delivered with a square-wave electroporation generator (CUY21EDIT; Nepa Gene). Uterine horns were returned into the abdominal cavity, and embryos continued their normal development.
3
Targeted In Utero Electroporation of Somatosensory Cortex
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IUE targeting the somatosensory cortex was performed using our previously published methods with minor modifications20 ,21 (link). Pregnant C57/BL6 mice were anesthetized at embryonic day 15 (E15) by intraperitoneal administration of a mixed solution of Ketamine HCl (100 mg/kg), Xylazine HCl (7.5 mg/kg), and Buprenorphine HCl (0.05 mg/kg). After the uterine horn was exposed by laparotomy, the shRNA plasmid (1 µg/µl) together with CAG promoter-driven GFP expression plasmids (1 µg/µl) (molar ratio, approximately 1:1) were injected into the lateral ventricles with a glass micropipette made from a micropillary tube (Narishige, Cat #GD-1), and electroporated into the VZ of the CP at E15. C11orf46 expression constructs (1 µg/µl) were also co-introduced for rescue experiments. For in vivo epigenomic editing, CAG-dCas9-ST (1 µg/µl), scFv-C11orf46 (1 µg/µl) and sgRNAs (1 µg/µl) together with shRNAs and GFP expression plasmid will be introduced at E15. For overexpression of Sema6a, CAG-Sema6a (2 µg/µl) or tdTomato (1 µg/µl) with GFP expression plasmid will be introduced at E15. For electroporation, Electrode pulses (40 V; 50 ms) were charged four times at intervals of 950 ms with an electroporator (Nepagene, Cat #CUY21EDIT). All experiments were performed in accordance with the institutional guidelines for animal experiments of Johns Hopkins University.
4
In utero Electroporation of Mouse Brains
5
In Utero Electroporation for Gene Delivery
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Cells were transfected in vivo through in utero electroporation, as described previously (Inoue et al., 2012 (link)). Briefly, plasmids carrying monomeric red fluorescent protein (mRFP) downstream of a CAG promoter (Addgene, MA, USA) were prepared using the EndoFree Plasmid Kit (Qiagen, Hilden, Germany). Pregnant mice and rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally) at E14.5 and E15.5, respectively, and their uterine horns were exposed. Plasmid DNA was dissolved in phosphate buffered saline (PBS) at a final concentration of 0.5 μg/μl with Fast Green (final concentration 0.05% [v/v]). Plasmids were injected into the lateral ventricle using a glass micropipette and a controlled pipette system (IM-30, Narishige, Tokyo, Japan). The micropipettes were generated from glass capillaries (outer diameter 1.0 mm; Harvard Apparatus, South Natick, MA, USA) that were pulled using a P-97 micropipette puller (Sutter Instrument Co., Novato, CA, USA). Electric pulses were produced by an electroporator (CUY21EDIT; NepaGene, Ichikawa, Japan) and delivered by a round plate forceps-type electrode with a 5-mm diameter (CUY650P5; NepaGene). Electric pulses (43 V, 50 ms) were applied five times at intervals of 950 ms. The uterine horns were then returned to the abdomen.
6
Overexpression of C7 Variants in Glioma Cells
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U251 glioma cells and HM cells were introduced from Kunming Cell Bank, Kunming Institute of Zoology, Chinese Academy of Sciences. The U251 cells were engineered to stably express mutant APP K670N/M671L (U251-APP) so that they would produce Aβ42 under doxorubicin induction [32 ,37 (link)]. We overexpressed wild type and mutant p.K420Q of C7 in these two cell lines to characterize their potential roles. Briefly, U251-APP cells were cultured in Roswell RPMI-1640 medium (HyClone, #C11875500BT) supplemented with 10% fetal bovine serum (Gibco-BRL; #10099–141), 100 U/ml penicillin and 100 mg/ml streptomycin in 5% CO2 at 37°C. HM cells were maintained in Dulbecco's modified Eagle's medium (Gibco-BRL; #11965–092) supplemented with 10% fetal bovine serum (Gibco-BRL; #10099–141). Transfection of empty vector (pReceiver-M14 [Cytomegalovirus promoter, 3 × Flag], GeneCopoeia, Inc.), or C7 wild type and mutant p.K420Q expression vectors, was performed using an electroporator (CUY21EDIT, Nepa gene Co., Japan) following the manufacturer's instructions. In brief, cells were trypsinized and washed three times with Opti-MEM medium (Gibco-BRL). Around 1 × 106 cells were resuspended in 100 μl Opti-MEM medium, and electroporated with 10 μg plasmids. Transfected cells were seeded in prewarmed growth medium for 72 h in 5% CO2 at 37°C before being harvested.
7
Electroporation-Assisted Transdermal Plasmid Delivery
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Skin was shaved at the site of injection. Each animal received id injections of 100 µl (1 mg/ml) of auxo-GTU®-multiHIV plasmid with or without EP. Phosphate-buffered saline (PBS) (100 µL) was injected as a control. EP was performed with a portable pulse generator (CUY21 EDIT; Nepa Gene, Ichikawa, Japan) and tweezer electrodes (6 pulses of 10 msec with output current 300–600 mA)51 (link). For imaging studies of antigen expression, 100 µl (250 µg) of auxo-GTU®-Luc-EGFP plasmid was injected.
8
In utero electroporation of optogenetic and fluorescent markers
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CAG:hM3Dq:IRES:GFP, CAG:hM3Dq:IRES:RFP or CamKII-ACE2N-mNeon-4AA were expressed in pyramidal cells via in utero electroporation. In utero electroporation was performed in E15.5 pregnant mice. Females were anaesthetised with isoflurane 2.5% before the abdomen was opened and the uterine horns exposed. DNA was delivered to the embryos via injection into the lateral ventricles with a glass micropipette. A total of 0.5-1 μL of DNA solution was injected into each embryo. The DNA solution contained Fast Green (0.3% (w/v)) and DNA in TE buffer (1500-2000 ng/ml). Five square electric pulses (30 V, 50 ms) were passed at 1 s intervals using a squarewave electroporator (CUY21EDIT; NEPA GENE). All successfully electroporated mice were included in all analyses.
9
In Utero Gene Electroporation in Mouse Embryos
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Plasmids were purified with the Endofree plasmid maxi kit (Sigma-Aldrich), injected in the lateral ventricle of E14.5 embryos in utero, and electroporated as previously described (Mire et al., 2012 (link)) using an electroporator (CUY21 Edit; Nepagene). Embryos were harvested in L15-glucose 48 h after electroporation. Brains were dissected and open or cut in the vibratome; the electroporated parts or the 100-µm sections were fixed in 10% ice-cold TCA for 20 min for pERM staining or in 4% PFA for 2 h for other stainings and processed for immunofluorescence as described previously (Deck et al., 2013 (link)).
10
In Utero Electroporation of Plasmids
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Plasmids were transfected by in utero electroporation using previously described methods.15 Briefly, E14 pregnant female C57BL/6 mice were anesthetized by an intraperitoneal injection of 2,2,2‐tribromoethanol (200–300 mg/kg body weight) prior to the experiments. A total of 1 μg of plasmid was injected by transuterine pressure microinjection into the lateral ventricle of the embryos by delivering five electrical pulses (40 V, 50 ms duration) at intervals of 950 ms using a square‐pulse electroporator (CUY21EDIT; Nepa Gene) and a tweezer‐type electrode with disc electrodes (5 mm in diameter) at the tip (CUY650‐5; Nepa Gene, Chiba, Japan). For the analysis of migration and cell shape, the brains of mice were fixed with 4% paraformaldehyde/0.1 M phosphate buffer (pH 7.4), cut coronally into 100 μm slices with a Vibratome (VT1000S; Leica Microsystems), and imaged using a laser‐scanning confocal microscope (LSM 5 PASCAL; Carl‐Zeiss).
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