In Utero electroporation (IUE) was performed as previously described (Saito and Nakatsuji, 2001 (link)) on E13.5 or E15.5 timed pregnant C57Bl/6 mice to label L5 or L2/3 PNs, respectively. The pCAG-GFP plasmid (Addgene #11150) was purified using the NucleoBond Extra Midi EF Kit (Clontech Laboratories). The plasmid was diluted to a final concentration of 1 μg/μl with sterile phosphate buffered saline (PBS) and colorized with 0.1% Fast Green (Sigma-Aldrich) dissolved at 37°C immediately prior to use. One to two microliters DNA plasmid was injected into the lateral ventricle (LV) through a pulled glass micropipette. Five pulses (25–30 V amplitude, 50 ms duration with 950 ms intervals) were delivered, targeting the motor or barrel cortex, using a platinum plate tweezers-type electrode connected to a square-pulse electroporator (CUY21, NEPA Gene).
Cuy21
Manufactured by Nepa Gene
Sourced in Japan
The CUY21 is a laboratory equipment designed for general scientific applications. It serves as a versatile tool for various research and testing purposes. The core function of the CUY21 is to provide a controlled environment for experiments and analyses, but a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Cuy650p3, by Nepa Gene (4 mentions)
Fast green, by Merck Group (3 mentions)
Pcaggs chr2 venus, by Addgene (2 mentions)
Mvx10, by Olympus (2 mentions)
Cuy650p5, by Nepa Gene (2 mentions)
Femtojet, by Eppendorf (2 mentions)
Somnopentil, by Kyoritsu Seiyaku (1 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (1 mentions)
Facsaria sorp, by BD (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
Lightcycler 96, by Roche (1 mentions)
M205 fa, by Leica (1 mentions)
Brdu solution, by Merck Group (1 mentions)
Stealth sirna, by Thermo Fisher Scientific (1 mentions)
Aav pcag flex egfp wpre, by Addgene (1 mentions)
Pegfp c1, by Takara Bio (1 mentions)
C57bl 6j mice, by Japan SLC (1 mentions)
Nepa21, by Nepa Gene (1 mentions)
Domitor, by Nippon Zenyaku Kogyo (1 mentions)
Vetorphale, by Meiji Seika Pharma (1 mentions)
27 protocols using cuy21
1
In Utero Electroporation of Mouse Cortex
2
ChR2-venus Expression in Visual Cortex
3
ChR2-venus Expression in Visual Cortex
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We expressed ChR2-venus in layer 2/3 pyramidal neurons over visual cortex via in utero electroporation onto C57Bl6 × CD1 mice at embryonic day 15.5. We used the offspring of a cross between CD1 females and C57BL/6 males (Charles River, UK), taking advantage of the fertility and fostering capability of CD1 females. Crossed mice had brown or black coats as described previously11 (link) and showed normal features in the pigmented epithelium of eye, confirmed with fundus images and sectioned images (data not shown). E15.5 timed-pregnant CD-1 mice were anesthetized with 2% isoflurane in oxygen. Up to 1 μl of DNA solution with Fast Green (Sigma, UK) was pressure-injected into left lateral ventricle of embryos. The solution2 (link),11 (link),21 (link) contained pCAGGS-ChR2-Venus (Addgene 15753, 1.5 μg/μl) and pCAG-mCherry (0.5 μg/μl). Electroporation was achieved with 5 square pulses (50 V, 50 ms, 1 Hz, CUY21, NepaGene, Japan). mCherry fluorescence was used to screen for positive animals at P0 under a fluorescent stereoscopic microscope (MVX10, Olympus). Images showing ChR2-venus expression in a whole brain in vivo and in sectioned slices are available in our previous study (Fig. 1d,e in Ref. 2 (link)).
Animals were maintained with a light-dark cycle of 12:12 h, and up to four mice were kept in one cage after weaning.
Animals were maintained with a light-dark cycle of 12:12 h, and up to four mice were kept in one cage after weaning.
4
In Utero CXCR5 Modulation in Mice
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Pregnant mice at E14.5 were anesthetized with 0.7% pentobarbital sodium. The abdomen was then sterilized, the uterus was exposed by incision, and a high concentration (2 µg/µL) of the CXCR5 knockdown plasmid, the knockdown control plasmid, the CXCR5 overexpression plasmid, or the overexpression control plasmid was injected into the lateral ventricles of intrauterine embryos through polished glass micropipettes. Then, CUY650P5 electrodes were placed on both sides of the fetal mouse head, and the positive electrode was placed tangentially in the target cortical area, followed by electroporation (CUY21, Nepa Gene, Ichikawa-City, Chiba, Japan) (pulse voltage: 33 V, pulse duration: 50 ms, pulse interval: 950 ms, number of pulses: 4). Finally, the uterus was returned to the abdominal cavity and the abdomen was sutured. After birth (E19/P0), brain slices were isolated and processed.
5
Multi-Lineage Tracing in Mouse Embryos
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GE-directed IUE was performed on E10.5 wild type, Ai9 reporter mice and Nxk2.1Cre; Ai9 mice as described17 (link)56 (link) with some modifications. Five electric pulses (27 V for 50 ms) were delivered at 950 ms intervals with forceps-shaped electrodes (CUY650P2, Unique Medical Imada) connected to an electroporator (CUY21, Nepa Gene).
To achieve labeling of all progenitor descendants, the Tol-2 system was used57 (link). For this purpose, 1–2 μg/μl pCAGGS58 (link)-Tol2 transposase (T2TP) and 1 μg/μl pT2K-CAGGS-enhanced green fluorescent protein (EGFP)22 (link) were co-electorporated with 1 μg/μl pCAGGS-tandem dimer tomato (tdTomato) or monomeric cherry (mCherry)59 to wild type and 1–2 μg/μl pCAGGS-T2TP and 1 μg/μl pT2K-CAGGS-EGFP to Nkx2.1Cre; Ai9 mice. As a second method to label all progenitor descendants, 1 μg /μl pCAGGS-Cre and 1 μg/μl pCAGGS-EGFP60 (link) were co-electroporated to Ai9 mice embryos. In either method, plasmids were dissolved in PBS.
In all samples, GFP and tdTomato fluorescence were enhanced by immunostaining against GFP and DsRed, respectively.
To achieve labeling of all progenitor descendants, the Tol-2 system was used57 (link). For this purpose, 1–2 μg/μl pCAGGS58 (link)-Tol2 transposase (T2TP) and 1 μg/μl pT2K-CAGGS-enhanced green fluorescent protein (EGFP)22 (link) were co-electorporated with 1 μg/μl pCAGGS-tandem dimer tomato (tdTomato) or monomeric cherry (mCherry)59 to wild type and 1–2 μg/μl pCAGGS-T2TP and 1 μg/μl pT2K-CAGGS-EGFP to Nkx2.1Cre; Ai9 mice. As a second method to label all progenitor descendants, 1 μg /μl pCAGGS-Cre and 1 μg/μl pCAGGS-EGFP60 (link) were co-electroporated to Ai9 mice embryos. In either method, plasmids were dissolved in PBS.
In all samples, GFP and tdTomato fluorescence were enhanced by immunostaining against GFP and DsRed, respectively.
6
In utero electroporation for gene knockdown
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For in utero electroporation44 (link),50 (link), pregnant ICR mice at embryonic day (E) 14–15 were deeply anesthetized with sodium pentobarbital (Somnopentil; Kyoritsu Seiyaku Co., Tokyo, Japan) injected intraperitoneally. Plasmids were dissolved in water. The pCAG plasmid vectors were used to knockdown the expression of CNTNAP2 or AHI1 in pyramidal neurons. The plasmids (0.5–2 µg/µl) were injected into either the left or the right lateral ventricle, or bilateral ventricles with a glass micropipette. Electric pulses (35 V for 50 ms, five to ten times at 950 ms intervals) were delivered through forceps-shaped electrodes (CUY650P5, Unique Medical Imada, Aichi, Japan) connected to an electroporator (CUY21, Nepa Gene, Chiba, Japan). Pups that expressed strong fluorescence in the prefrontal cortex of both hemispheres were used for behavior experiments.
7
In Utero Electroporation of Murine Brain
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Timed-pregnant mice were deeply anesthetized with isoflurane. Plasmid DNA (1–2 µg/µl, pCX-EGFP) was microinjected into the third or lateral ventricles of E12.5 brains and electroporated using an electroporator (Fig. 4A ; five 50 millisecond pulses of 50 V for the TE or 40 V for the medial cortex, with an interval of 950 milliseconds; CUY21, NEPA GENE) with a forceps-type electrode (CUY650P3). Embryos or postnatal mice were dissected 48 hours to 2 weeks after electroporation. All data were obtained from at least three independent experiments.
8
Quantitative RT-PCR Analysis of Nmnat Knockdown
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Total RNA was purified from mouse retinas using Sepasol RNA I Super G (Nacalai Tesque, Japan), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Japan). A quantitative polymerase chain reaction (qPCR) was performed by the SYBR Green-based method with the Roche Light Cycler 96 (Roche Diagnostics, Japan). Gapdh was used as an internal control, and primer sequences were as previously reported (Kuribayashi et al., 2018 (link)). To examine the knockdown efficiency of shRNA-expressing plasmids, a total of 100 μg of plasmids containing 80 μg of pU6-shNmnat2 or pU6-Scramble and 20 μg of enhanced green fluorescent protein (EGFP)–expressing plasmids (pCAG-EGFP) were electroporated into NIH3T3 cells with electroporator CUY-21 (Nepa Gene, Japan). After 1–2 d, NIH3T3 cells were treated with trypsin and filtered, and EGFP-positive cells were collected by a cell sorter, FACS Aria SORP (BD Biosciences, USA). Total RNA was purified from the sorted cells, and cDNA was synthesized. Knockdown efficiency of shNmnat2 was examined by qPCR using primer for Nmnat2. Effects of shNmnat2 for the expression of Nmnat1 and Nmnat3 were also examined by RT-qPCR using primers for Nmant1 or Nmnat3.
9
In utero electroporation for gene overexpression
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In utero electroporation was performed as previously described (Saito and Nakatsuji, 2001 (link)). Briefly, DNA plasmids were mixed with Fast Green and injected into the fourth ventricle of embryonic brains from outside the uterus with a glass micropipette. Holding the embryo in utero with forceps-type electrodes (NEPA GENE), 50 ms of 40 V electronic pulses were delivered five times at intervals of 950 ms with a square electroporator (Nepa Gene, CUY21). The plasmids used in this study are displayed in the Key Resource table. The primer sequences to clone to clone the mouse Olig3 (NM_053008.3) and Pax2 (NM_011037.5) genes are displayed in in the Key Resource table. The electroporated plasmid DNA mixtures were as follows: (i) for the control experiment, pCAG-GFP (0.5 mg ml−1) + pCAG-Empty-IRES-GFP (0.5 mg ml−1); (ii) for the Olig3 overexpression experiment, pCAG-Olig3-IRES-GFP (0.5 mg ml−1); and (iii) for the Pax2 overexpression experiment, pCAG-Pax2-IRES-GFP (0.5 mg ml−1).
10
In vivo Electroporation of Embryonic Mouse Brains
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Timed-pregnant mice were deeply anesthetized with isoflurane. Plasmid DNA (1–2 µg/µl) was microinjected into the lateral or third ventricles of embryonic brains and electroporated using an electroporator (Fig. 2A , Fig. 5A ; five 50 millisecond pulses of 40 V for E12.5 embryos, with an interval of 950 milliseconds; CUY21, NEPA GENE) with a forceps-type electrode (CUY650P3). Mouse embryos were dissected 5–6 days after electroporation. All data were obtained from at least three independent experiments.
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