Synthetic crRNA and trans-activating crRNA (tracrRNA) were obtained from Fasmac Co., Ltd. (Kanagawa, Japan). The sequences of the two crRNAs for amhr2 exons were as follows: Target 1, CAGUGACAGCAUCCAGCUGGguuuuagagcuaugcuguuuug; Target 2, UGCUUUGGGACUGCUGCUGUguuuuagagcuaugcuguuuug. These sequences were designed as described previously [25 (link)]. The two crRNAs (250 ng/µL), tracrRNA (500 ng/µL), and Cas9 protein (750 ng/µL; Nippon Gene) were mixed and injected into fertilized eggs immediately after fertilization using a microinjector (IM-9B; Narishige, Tokyo, Japan) as described previously [25 (link)]. The two crRNAs and Cas9 protein without tracrRNA were injected for the experimental control group. The artificial fertilization, microinjection, and rearing of the experimental fish were performed at Nansei Field Station, Japan Fisheries Research and Education Agency. Eggs and sperm were produced by 3-year-old females and males.
Im 9b
Manufactured by Narishige
Sourced in Japan
The IM-9B is a microinjector produced by Narishige. It is a manual device used for the controlled injection of liquids or gases into biological samples, such as cells or tissues, during microscopic-scale procedures. The IM-9B provides precise control over the volume and rate of injection.
Automatically generated - may contain errors
Lab products found in correlation
7 protocols using im 9b
1
CRISPR/Cas9 Mediated Genome Editing in Teleosts
2
Assessing Brain Tissue Proliferation
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The protocol to assess proliferation ability of brain tissues was originally set up in the lab of Cayetano Gonzalez [41 (link)]. Briefly, a central brain fragment (expressing a fluorescent probe) was transplanted in the abdomen of a live wild-type adult female fly and the growth of the transplant was monitored over time. Control or mutant third instar larval brains, expressing either His2Av::GFP or UAS-Cherry::Tubulin under the control of Insc-Gal4 were dissected in sterile PBS (Sigma). One brain lobe was transplanted in the abdomen of a wild-type adult fly using a pulled capillary with a beveled tip (around 150 μm diameter) adjusted to a microinjection system (IM-9B; Narishige). After transplantation, host flies were maintained at 18°C overnight and transferred at 25°C for a month. Tumor growth was monitored every one or two days upon 30 days for the appearance of a mCherry or GFP signal in the abdomen. For aurA and strong polo mutants, transplant growth was assessed over a 15-days period because these tumors were particularly aggressive and killed the host flies before 30 days. The neuroblast-specific Insc-Gal4 was used to assay the effect of PoloT182D::GFP variant for tumor growth.
3
Intracranial Administration of Kisspeptin Peptides
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Central administration of Kiss1 peptides was performed as described previously in Kizil and Brand (2011) and Ogawa et al. (2012)10 (link),64 (link). For whole brain gene expression assay, AB fish were anesthetized by immersion in 0.01% benzocaine (Sigma, USA) solution and intracranially administered with 1 μL of either kisspeptin1-15 (Kiss1; pyroglut-NVAYYNLNSFGLRY-NH2; Open Biosystems) or kisspeptin2-10 (Kiss2; FNYNPFGLRF-NH2; BioGenes) with two different doses (10–12 and 10–9 mol/fish in distilled water). For injection, the cranial bone close to the habenula above the left side of the anterior part of the optic tectum was incised using a sterilized barbed-end needle (BD Precision Glide 30G × 0.5″, BD Medical, New Jersey) (Fig. 1 A), and through this incision,
Kiss1 solution was administered using a heat-pulled glass capillary micropipette attached with a microinjector (IM-9B, Narishige, Tokyo, Japan) (Fig.1 B). For control, the same volume of distilled water was administered. Fish in sham group received the puncture procedure without any administration.
Kiss1 solution was administered using a heat-pulled glass capillary micropipette attached with a microinjector (IM-9B, Narishige, Tokyo, Japan) (Fig.
4
Modulation of Morphine-Induced Fear Memory
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To examine the effect of Kiss1 on morphine-induced fear memory impairment, fish (Group 4, Fig. 4 B) were administered with Kiss1. Intracranial administration of Kiss1 was carried out as described previously44 (link). Briefly, fish were anesthetized by immersion in 0.01% benzocaine (Sigma, USA) solution and placed on a sponge soaked with water. The cranial bone at the telencephalon-diencephalon border, close to the habenula above the left side of the anterior part of the optic tectum was incised using a sterilized barbed-end needle (30G × 1″ Terumo). Then, through this incision, the fish was intracranially injected with 1 µl of zebrafish kisspeptin1-15 (pyroglut-NVAYYNLNSFGLRY-NH2; Open Biosystems) at the dose of 10–21 mol/fish or MQ water (control) by heat-pulled glass capillary micropipette (inner diameter: 1 mm, model G-1, Narishige, Japan) attached with microinjector (IM-9B; Narishige). The dose of Kiss1 utilized in the present study (10–21 mol/fish in 1 µl) was chosen based on previous study46 (link), which has been demonstrated to depolarize ventral habenula neurons in zebrafish.
5
Forming Planar Lipid Bilayers with Nanopores
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The contact
bubble bilayer (CBB) method33 (link),34 (link) was performed under
an inverted microscope (IX71; Olympus, Tokyo,
Japan), and images were recorded using a digital camera (MS-200; Bio
Craft, Tokyo, Japan). Glass pipettes for bubble formation and perfusion
inside the bubble were fabricated by pulling a borosilicate glass
capillary (BF100-50-10; OD/ID; 1.0/0.5 mm, Sutter Instrument, Novato,
CA) with a micropipette puller (PC-100; Narishige, Tokyo, Japan).
Then, the tips of the pipettes were cut to obtain a tip diameter of
around 30 μm and lightly polished using a micro-forge (MF-900;
Narishige). First, 10 mg/mL DPhPC solution (in n-decane)
(100 μL) was put on a slide glass. Next, two glass pipettes,
one filled with the buffer solution with 100 nM αHL and the
other filled with the buffer solution containing 100 nM target DNA
were immersed into the DPhPC solution. Then, two water bubbles were
formed from the pipettes by applying pressure inside the pipettes.
Finally, the pBLM was formed by manipulating the pipettes so the two
water bubbles came into contact. The pipette position was controlled
by micromanipulators (UM-3C; Narishige) under the microscope. The
pressure in the pipette was regulated by a micro injector (IM-9B;
Narishige). Ag/AgCl electrodes were placed in both glass pipettes.
The ionic current was measured by applying a transmembrane potential
of +180 mV.
bubble bilayer (CBB) method33 (link),34 (link) was performed under
an inverted microscope (IX71; Olympus, Tokyo,
Japan), and images were recorded using a digital camera (MS-200; Bio
Craft, Tokyo, Japan). Glass pipettes for bubble formation and perfusion
inside the bubble were fabricated by pulling a borosilicate glass
capillary (BF100-50-10; OD/ID; 1.0/0.5 mm, Sutter Instrument, Novato,
CA) with a micropipette puller (PC-100; Narishige, Tokyo, Japan).
Then, the tips of the pipettes were cut to obtain a tip diameter of
around 30 μm and lightly polished using a micro-forge (MF-900;
Narishige). First, 10 mg/mL DPhPC solution (in n-decane)
(100 μL) was put on a slide glass. Next, two glass pipettes,
one filled with the buffer solution with 100 nM αHL and the
other filled with the buffer solution containing 100 nM target DNA
were immersed into the DPhPC solution. Then, two water bubbles were
formed from the pipettes by applying pressure inside the pipettes.
Finally, the pBLM was formed by manipulating the pipettes so the two
water bubbles came into contact. The pipette position was controlled
by micromanipulators (UM-3C; Narishige) under the microscope. The
pressure in the pipette was regulated by a micro injector (IM-9B;
Narishige). Ag/AgCl electrodes were placed in both glass pipettes.
The ionic current was measured by applying a transmembrane potential
of +180 mV.
6
Trout Germ Cell Transplantation
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ASGs derived from 12- to 15-month-old dominant orange-colored (heterozygous, orange/black; OR/wt) vasa-gfp transgenic trout (heterozygous, vasa-gfp/−) were cultured and used for transplantation assays. Approximately 15,000 ASGs from cultured or freshly dispersed testicular cells were microinjected into the peritoneal cavity of each nontransgenic triploid hatchling (30 days old) by using a microinjector (IM-9B, Narishige) under a stereomicroscope (SZX-10, Olympus)11 (link). In each trial, testes from more than 10 individual fish were pooled and used to avoid individual variation in donor trout. Some of the recipients were dissected and their gonads were observed under a fluorescence microscope at 20, 70, and 139 dpt. The percentage of recipients possessing incorporated ASGs in their genital ridges (transplantation efficiency) and the total number of incorporated ASGs in each recipient were determined at 20 dpt.
7
CRISPR-mediated Knockdown of amh Gene
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Synthetic CRISPR RNAs (crRNAs) and trans-activating crRNA (tracrRNA) were obtained from Fasmac Co., Ltd. (Kanagawa, Japan). The sequences of the two crRNAs for amh were Target 1 5′-CUAUCUGCAGCUCGUAGGUAguuuuagagcuaugcuguuuug-3′ and Target 2 5′-AAUUAAAAAGCACAUUUUGaguuuuagagcuaugcuguuuug-3′. These sequences were designed on the basis of Sawamura et al. (Sawamura et al., 2017 (link)). Two crRNAs (250 ng/μl), tracrRNA (500 ng/μL), and Cas9 protein (750 ng/μl; Toyama, Nippongene) were mixed and immediately injected into fertilized eggs using a microinjector (IM-9B, Narishige, Tokyo, Japan). Two crRNAs and Cas9 protein without tracrRNA were injected as experimental controls.
The artificial fertilization, microinjection, and rearing of the experimental fishes were performed at the Nansei Field Station, Japan Fisheries Research and Education Agency. Eggs and sperm from three-year-old females and males were used. They were mixed in a plastic beaker sampler and activated with sterilized seawater (18°C) for artificial fertilization. At 200 dah, the gonads were isolated from each fish; one lobule was processed for histological analysis and the other was used for sex genotyping. All the fish were sampled after ensuring that they had been completely euthanized by an overdose of 2-phenoxyethanol (Wako Chemicals).
The artificial fertilization, microinjection, and rearing of the experimental fishes were performed at the Nansei Field Station, Japan Fisheries Research and Education Agency. Eggs and sperm from three-year-old females and males were used. They were mixed in a plastic beaker sampler and activated with sterilized seawater (18°C) for artificial fertilization. At 200 dah, the gonads were isolated from each fish; one lobule was processed for histological analysis and the other was used for sex genotyping. All the fish were sampled after ensuring that they had been completely euthanized by an overdose of 2-phenoxyethanol (Wako Chemicals).
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