Pregnant mice were ethically anesthetized with isoflurane combined with oxygen (TEM SEGA). The mice were coinjected with pCAG-GFP and pCAG expressing different constructs of Reg-1α (1.5 μg/μl) in a 1:3 ratio, were mixed with 0.5 μl Fast Green (0.03%, Sigma) and injected (2 μl) into the lateral ventricle of E14.5 embryonic brains using a pulled glass micropipette. Electrode pair (5 mm in diameter; CUY650-P5, NEPA Gene) attached to an ECM 830 electroporator (BTX Harvard Apparatus) transmitted 5 squares 40 V electric pulses for 50 ms at 950 ms intervals through the uterine wall. Each condition was applied to at least three independent litters, targeting the same dorso-lateral region of the neocortex. For 5-bromo-2-deoxyuridine labeling (BrdU, Sigma), electroporated pregnant dams were intraperitoneally (i.p) injected with BrdU (100 μg/g) 1 day after electroporation and sacrificed 24 h later. The sections were stained for rabbit anti-T-box brain 1 (Tbr1) to delineate the cortical plate. The percentage of targeted cells was determined by dividing the number of GFP positive cells in each layer (cortical plate and intermediate zone/ventricular zone) by the total number of GFP-positive cells in the entire section.
Ecm 830 electroporator
Manufactured by Harvard Apparatus
Sourced in Japan, United States
The ECM 830 electroporator is a laboratory instrument designed for the delivery of electric pulses to cells or tissues. It is used to temporarily increase the permeability of cell membranes, allowing the introduction of various molecules, such as DNA, RNA, or drugs, into the cells. The ECM 830 provides a controlled and adjustable electric field to facilitate this process, known as electroporation.
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Lab products found in correlation
Fast green, by Merck Group (5 mentions)
Bovine placental hyaluronidase, by Merck Group (4 mentions)
Platinum tweezertrodes, by Harvard Apparatus (2 mentions)
Shisa7 s405a gfp, by Merck Group (2 mentions)
Cuy21 electroporator, by Nepa Gene (2 mentions)
C57bl 6 and cd1, by Taconic Biosciences (2 mentions)
Swiss webster, by Taconic Biosciences (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Cuy650p5, by Nepa Gene (1 mentions)
Optimem without phenol red, by Thermo Fisher Scientific (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
X vivo medium, by Lonza (1 mentions)
Nhei hf, by New England Biolabs (1 mentions)
Bamhi hf, by New England Biolabs (1 mentions)
Oligonucleotide library, by Agilent Technologies (1 mentions)
Q5 polymerase, by New England Biolabs (1 mentions)
Mpc 200, by Sutter Instruments (1 mentions)
28 protocols using ecm 830 electroporator
1
In Utero Electroporation of Embryonic Mouse Neocortex
2
Stable Transgene Expression via PiggyBac
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We utilized the PiggyBac transposon system to allow stable transgene expression throughout life. Briefly, 0.5–1 µl of plasmid DNA (1 µg/µl) was injected into the right lateral ventricle of E15.5 CD1 embryos and electroporated into the S1 cortical neuroepithelium using five 100 ms pulses at 30 V delivered by external 3 mm paddle electrodes (Nepagene, Chiba, Japan) clamped across the uterus, which are connected to an ECM 830 electroporator (BTX Harvard Apparatus, Holliston, MA, USA). Plasmid expression constructs were pCAG-PBase and pPBCAG-GFP (kind gifts from Joseph LoTurco), pPBCAG-hM3Dq (subcloned from pcDNA5/FRT-HA-hM3D(Gq); Addgene #45547; into the pPBCAG vector backbone), and pCAG-Kir2.1 (kind gift from Yoshiaki Tagawa). Control GFP mice were electroporated with 1:1 ratio of pCAG-PBase and pPBCAG-GFP. hM3Dq/GFP mice were electroporated with 1:1:1 ratio of pPBCAG-hM3Dq, pCAG-PBase, and pPBCAG-GFP. Kir2.1/GFP mice were electroporated with a 1:1:1 ratio of pCAG-Kir2.1, pCAG-PBase, and pPBCAG-GFP. The E15.5 embryos of pregnant CD1 dams were electroporated at The University of Queensland and, following birth, the pups were transported to The University of Melbourne between P10 and P20 for CNO administration and further experimentation and analysis.
3
Silencing SOX2 in Neurospheres
4
In Vivo Electroporation in Mice
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The electroporation was performed on mice anesthetized with ketamine (150 mg/kg)/xylazine (10 mg/kg) cocktail, each time injecting 25 µg of plasmid DNA. After plasmid injection, the TA muscle was electroporated with 10 pulses of 180 V/cm current at 1 s interval between the consecutive pulses. For the procedure the ECM-830 electroporator (catalog No. 45-0052INT, BTX Harvard Apparatus, Holliston, MA, USA) was used. The experiment was repeated three times, each time using a new mouse so in total three P7 and three P60 mice were used. All mice were male. approved by the Ethics Committee of Nencki Institute of Experimental Biology (protocol code 629/2018 22.05.2018).
5
Quantifying NIS Expression in Electroporated Cells
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2 × 106 MC38 or TC-1 cells were washed twice in X-vivo medium (Lonza) and electroporated with 20 µg NIS-RNA in 4-mm cuvettes using ECM 830 electroporator (BTX Harvard Apparatus) (300 V for MC38, 260 V for TC-1, 1 pulse, 15msec for both cell lines). Cells were harvested 6, 24 and 48 h after electroporation and stained with AlexaFluor 647 labeled-human NIS antibody (1:100 dilution) for 30 min at 4 °C in the dark. Live-dead staining was performed using Fixable Viability Dye eFluor 780 (1:3000 dilution) (Thermofisher Scientific). Data were acquired with FACS Canto II flow cytometer (BD Biosciences) and analyzed by FlowJo Software ver 10.4 (Tree Star). % NIS + cells as well as mean fluorescence intensity (MFI) of NIS expression of live cells was presented. Cells electroporated without RNA served as mock control.
6
Chicken Embryo Electroporation for Gene Delivery
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ECM 830 electroporator (Harvard Apparatus®) was used to generate square-wave electric pulses. The head of the embryo was exposed by cutting the chorionic membrane. A solution (20 mM HEPES, 135 mM KCl, 2 mM MgCl2, 0.5% Ficoll 400, 2 mM ATP/5 mM glutathione) containing the pGeneClip-miRNA plasmid (20 ng/μl) was injected into the canal of the neural tube under illumination in a surgical microscope (Leica M320). Platinum Tweezertrodes (5 mm, Harvard Apparatus®) were carefully positioned bilaterally around the embryo’s head and 4 mm apart. Parameters for ex ovo electroporation of chicken embryos were adapted from Sauka–Spengler et al. Electroporation was carried out at 62.5 V/cm, 50 ms, 5 pulses at 1-s intervals. The electroporated embryos were then incubated for 24 h at 37 °C before harvesting for histological processing.
7
Electroporation of Chorioallantoic Membrane
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ECM 830 electroporator (Harvard Apparatus®) was used to generate square-wave electric pulses. The solution (20 mM HEPES, 135 mM KCl, 2 mM MgCl2, 0.5% Ficoll 400, 2 mM ATP/5 mM glutathione) containing the pGeneClip-miRNA plasmid (20 ng/μl) was loaded on top of the chorioallantoic membrane and gold-plated Genetrodes (3 mm L-Shape, Harvard Apparatus®) were placed on either side of the folded membrane. Electroporation was carried out at 62.5 V/cm, 50 ms, 2 pulses at 1-s intervals. The electroporated embryos were then incubated for 24 h at 37 °C before capturing the photograph followed by harvesting the membrane for histological processing.
8
Constructing AT118-H Nanobody Library
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The library of AT118-H variants was constructed by two-step-overlap-extension PCR. A set of ten primers (P94-P103) encoding mixed codons at seventeen amino acid positions was mixed in equimolar ratios (Integrated DNA Technologies, Supplementary Table 6 ). The mixed primer stock (25-50 nM final concentration) was amplified with Phusion polymerase to create the full length nanobody library DNA. AT118-H library DNA was successively amplified and homology arms corresponding to the pYDS649 plasmid encoding nourseothricin (nat) resistance (pYDS2.0) were added with primers P104-pYDSRev1, pYDSFwd2-pYDSRev2, pYDSFwd3-pYDSRev2. 37.5 μg of YDS2.0 digested with NheI-HF and BamHI-HF (New England Biolabs) and 187.5 μg nanobody insert DNA were transformed into 7.5E9 BJ5465 yeast with a 96-well ECM 830 Electroporator (BTX-Harvard Apparatus). Yeast were recovered in YPAD for 1.5 hours. Transformed cells containing the nanobody plasmid were selected with the addition of nat. Dilutions of transformed yeast were plate on YPAD + nat as single colonies to estimate the library diversity.
9
Ex Ovo Electroporation of Chicken Embryos
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ECM 830 electroporator (Harvard Apparatus®) was used to generate square-wave electric pulses. A solution (20 mM HEPES, 135 mM KCl, 2 mM MgCl2, 0.5% Ficoll 400, 2 mM ATP/5 mM glutathione) containing the molecular beacons (200 nM) was injected into the canal of the neural tube under illumination using a surgical microscope (Leica M320). Platinum Tweezertrodes (5 mm, Harvard Apparatus®) were carefully positioned bilaterally around the embryo’s head and 4 mm apart. Parameters for ex ovo electroporation of chicken embryos were adapted from Sauka–Spengler et al. [81 (link)]. Electroporation was carried out at 62.5 V/cm, 50 ms, 5 pulses at 1-s intervals. The electroporated embryos were then incubated for 24 h at 37 °C before harvesting for histological processing.
10
In Utero Electroporation for Cortical Neurogenesis
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Time-mated CD1 pregnant dams were used at S20 (E12, deep layers neurogenesis in the neocortex) or S23 (E15, upper layers neurogenesis) for all experiments. Deep anesthesia was achieved as described above. Following exposure of the uterine horns via laparotomy, various combinations of plasmids (Supplementary Table 1 ) were microinjected in the lateral ventricle with a picospritzer II holding a glass pulled pipette. The plasmids were then electroporated into the right primary somatosensory cortex (S1) with 3 mm diameter microelectrodes (Nepagene) delivering 5 (100 ms, 1 Hz) approximately 36 V square wave pulses from an ECM 830 electroporator (BTX Harvard Apparatus). Once this procedure was completed for each embryo, the uterine horns were replaced inside the abdominal cavity and the incision was sutured closed. Animals were then subcutaneously injected with 1 ml of Ringer’s solution and recovered in a humidified chamber at 28 °C. For analgesia, buprenorphine (0.05 mg/kg) and meloxicam (5 mg/kg) were injected subcutaneously, and edible buprenorphine was injected into jelly and placed in the cage.
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