Pregnant E13.5 mice were anaesthetized by intraperitoneal administration of dexmedetomidine (0.3 mg/kg; Zenoaq, Tokyo, Japan), midazolam (2 mg/kg; Astellas Pharma, Tokyo, Japan), and butorphanol tartrate (2.5 mg/kg; Meiji Seika Pharma, Tokyo, Japan) on a heating pad. The uterine horns were exposed, and approximately 1 μl of plasmids mixed with Fast Green (Sigma-Aldrich, St. Louis, MO) in TE buffer was manually injected into the developing aqueduct using a pulled glass micropipette (G-1.0; Narishige, Tokyo, Japan). Concentrations were as follows: pNeuroD-Cre (0.25 μg/μl), pCAG–FloxP–mKO2-F (0.25 μg/μl), pCAG-Dscam-mEGFP (2 μg/μl), pCAG-H2B-EGFP (BFP) (0.25 μg/μl), pmU6-shRNA constructs (1 μg/μl), pCAG-EGFP (0.25 μg/μl), pCAG-resDscam (2 μg/μl), and pCAG-3xFlag-RapGEF2 (2 μg/μl). Electric pulses (33 V, four pulses; 30 ms on, 970 ms off) were delivered across the heads of the embryos targeting the dorsal-medial part of the midbrain with forceps-type electrodes (CUY650P5; Nepa Gene, Ichikawa City) connected to an electroporator (CUY21E, Nepa Gene). After electroporation, uteri were placed back in the abdominal cavity, allowing embryos to continue developing. All surgical procedures were completed within 30 min, after which the mice recovered on a heating pad for 30 min. After 2 or 5 days, embryos were collected and subjected to immunohistochemical analysis.
Cuy21e
Manufactured by Nepa Gene
Sourced in Japan
The CUY21E is a laboratory equipment designed for use in genetic research and analysis. It serves as a compact and efficient instrument for performing various tasks related to DNA and RNA manipulation. The core function of the CUY21E is to facilitate the controlled and precise delivery of substances, such as buffers or reagents, into cells or tissues for the purpose of gene expression studies or other experimental procedures.
Automatically generated - may contain errors
8 protocols using cuy21e
1
In Utero Electroporation of Murine Midbrain
2
In Vivo Embryonic Brain Manipulation
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Pregnant mice were maintained under inhaled isoflurane anaesthesia for the duration of the procedure. The uterine horns were exposed. In some experiments, 1 to 2 μl of Vismodegib (5 mM solution in DMSO; Stratech) or DMSO alone was injected into the lateral ventricle of each embryo’s brain with a glass micropipette. In other experiments, plasmids were injected into the lateral ventricle of each embryo’s brain at 1 to 4 mg mL−1, the embryo in the uterus was placed between tweezer-type electrodes (CUY650; Nepagene), and an electroporator (CUY21E; Nepagene) was used to deliver 6 pulses (30 V, 50 ms each, 950 ms apart). In both cases, the uterine horns were replaced, the abdominal wall was sutured, and animals recovered. Processing was as described above.
Plasmids used in this study were as follows: CAG-FoxG1-IRES-mCherry (kindly provided by Goishi Myioshi, Tokyo Women’s Medical University, Japan); Scrambled shRNA control in pGFP-V-RS shRNA Vector (TR30013, Origene); Smoothened shRNA in pGFP-V-RS shRNA Vector (TG510788, Origene).
Plasmids used in this study were as follows: CAG-FoxG1-IRES-mCherry (kindly provided by Goishi Myioshi, Tokyo Women’s Medical University, Japan); Scrambled shRNA control in pGFP-V-RS shRNA Vector (TR30013, Origene); Smoothened shRNA in pGFP-V-RS shRNA Vector (TG510788, Origene).
3
In Utero Electroporation of Mouse Embryos
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Pregnant dams from wild-type (WT) ICR, Prdm8-mVenus, or Prdm8−/− mice were anesthetized by intraperitoneal injection with pentobarbital. Two uL of a mixture of plasmid DNA, which include 2.5 mg/mL target plasmid, 0.8 mg/mL reporter plasmid, and 2 mg/mL Fast Green was directly injected into the lateral ventricles of the embryonic forebrain by using a glass micropipette. In the case of conditional expression vector, a mixture of plasmid, which include 2.5 mg/mL target plasmid, 1 mg/mL pCAG-FloxP-EGFP-N1, 0.3 µg/mL pCAG-Cre, and 2 mg/mL Fast Green was used. The electroporation was performed using an electroporator (CUY21E, Nepa Gene) as previously described [34] (link), [35] (link).
4
In utero Electroporation for Gene Delivery
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In utero electroporation was performed as described previously [26 (link),27 (link),28 (link)]. Briefly, the Institute of Cancer Research (ICR) pregnant mice with E15.5 embryos were anesthetized, and the uterine horns were exposed. Approximately 1 μL of 1 μg/μL pAAV-H1-hSyn-EGFP (Control)/pAAV-H1-shIQSEC2-hSyn-EGFP (IQSEC2-KD) plasmid DNA solution was injected together with 0.01% Fast green for visualizing delivery into the lateral ventricles of the embryos. The embryos were then subjected to a 5-time-repeat of square electric pulses (35 V, 50 ms, 1 Hz) using an electroporator (CUY21E; NEPA Gene, Chiba, Japan) and returned to the uterus for normal maturation. After delivery, positively transfected pups were screened by EGFP signals through the scalp using a blue LED handy light with a filter. On postnatal day 14–19, morphologically normal brains were taken for electrophysiology and histology.
5
In utero electroporation of mouse embryos
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In utero electroporation was performed essentially as described previously [35 (link), 36 (link)]. Briefly, pregnant Institute of Cancer Research mice at E15.5 were anesthetized, and the uterine horns were exposed. Approximately 1 μl of DNA solution was injected into the lateral ventricles of embryos. The embryos were subjected to five square electric pulses (35 V, 50 ms, 1 Hz) using an electroporator (CUY21E; NEPA Gene). The brains with abnormal morphology were excluded from the experiments.
6
Neonatal In Vivo Electroporation for CA-AhR Expression
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Neonatal in vivo electroporation was performed as described previously34 (link), followed by cellular migration and morphology analyses. On PND 1, male pups were deeply anesthetized by hypothermia (30 s on crushed ice). The plasmid solution was prepared as described and used for neonatal in vivo electroporation to induce CA-AhR expression in cells in the RMS, OB, and AOB. For neonatal electroporation, approximately 0.5–1.0 μl of plasmid solution was injected into the lateral telencephalon ventricle of each pup using a hand-made glass micropipette (GD-1; Narishige, Tokyo, Japan), followed by electroporation using a tweezer-type electrode (CUY650-5; Tokiwa Science, Fukuoka, Japan) and an electroporator (CUY21E; Nepa gene, Chiba, Japan). Electric pulses (99 V; 50 ms) were charged four times at 950-ms intervals. Pups electroporated with pCAGGS1-EGFP and pCAGGS1-EGFP + pCAGGS1-CA-AhR were designed as the control and CA-AhR groups, respectively.
7
In utero Electroporation for Embryonic Gene Manipulation
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In utero electroporation was performed as essentially described previously15 (link). Briefly, pregnant ICR mice at E15.5 or E13.5 were anesthetized, and the uterine horns were exposed. Approximately 1 μl of DNA solutions containing 0.01% fast green were injected into the lateral ventricles of embryos using a pulled borosilicate glass capillaries (B120F-4; World Precision Instruments). The DNA solutions contained 1.5 μg/ul Cas9/sgRNA plasmid, 1.5 μg/ul pBSSK-EGFP-β-actin-donor, and 1.0 μg/ul pCAG-TagRFP or 1.0 μg/ul pCAG-EGFP-β-actin and 1.0 μg/ul pCAG-TagRFP. The head of embryo in the uterus was placed between tweezers-type electrodes with 5 mm diameter (CUY650P5; NEPA Gene). Each injected embryos was subjected to five square electric pulses (35 V, 50 msec, 1 Hz) using electroporator (CUY21E; NEPA Gene). After electroporation, the embryos were returned to the abdominal cavity to allow continuous development. Transfected pups were identified at P0-2 by TagRFP signals through scalp using a fluorescence stereomicroscope. The animal protocol was approved by the Animal Care and the Use Committee of Shinshu University. The methods were carried out in accordance with the Regulations for Animal Experimentation of Shinshu University.
8
In Utero Electroporation of Murine Cerebellum
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Pregnant E12.5 mice were anesthetised by intraperitoneal administration of dexmedetomidine (0.3 mg/kg; Zenoaq, Tokyo, Japan), midazolam (2 mg/kg; Astellas Pharma, Tokyo, Japan), and butorphanol tartrate (2.5 mg/kg; Meiji Seika Pharma, Tokyo, Japan) on a heating pad. The uterine horns were exposed, and ~1 μL plasmids mixed with Fast Green (Sigma-Aldrich) in TE buffer were manually injected into the developing aqueduct using a pulled glass micropipette (G-1.0; Narishige, Tokyo, Japan). Concentrations were as follows: 0.25 μg/μL pCAG–hyPBase, 0.25 μg/μL pPB-CAG-DsRed2, and 1.5 μg/μL pCAG–Dscam–mEGFP. Electric pulses (33V, four pulses; 30 ms on, 970 ms off) were delivered across the heads of the embryos targeting the dorsal-medial part of the cerebellum with forceps electrodes (CUY650P5; Nepa Gene, Ichikawa City, Japan) connected to electroporator (CUY21E; Nepa Gene). After electroporation, uteri were placed back into the abdominal cavity, allowing embryos to continue developing. All surgical procedures were completed within 30 min, after which the mice were recovered on a heating pad for 30 min. P15 mouse brains were fixed and subjected to immunohistochemical analysis.
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