All reporter plasmids were integrated into a unique landing site on the third chromosome using the VK00033 strain (60 (link)). PhiC31 was maternally provided using the vas-phiC31 strain (61 (link)). Microinjection was performed as previously described (62 (link)). In brief, 0–1 h embryos were collected and dechorionated with bleach. Aligned embryos were dried with silica gel for ∼7 min and covered with FL-100-1000CS silicone oil (Shin-Etsu Silicone). Subsequently, microinjection was performed using FemtoJet (Eppendorf) and DM IL LED inverted microscope (Leica) equipped with M-152 Micromanipulator (Narishige). Injection mixture typically contains ∼500 ng/μl plasmid DNA, 5 mM KCl, 0.1 mM phosphate buffer, pH 6.8. The mini-white marker was used for screening.
M 152 micromanipulator
Manufactured by Narishige
Sourced in Japan
The M-152 Micromanipulator is a precision instrument designed for the accurate and controlled manipulation of microscopic objects. It provides precise three-dimensional movement and positioning of attached tools or samples under a microscope. The core function of the M-152 is to enable the user to delicately and precisely control the movement and positioning of small-scale samples or instruments during microscopic procedures.
Automatically generated - may contain errors
Lab products found in correlation
Dmil led inverted microscope, by Leica (5 mentions)
Femtojet, by Eppendorf (5 mentions)
Bf100 50 10, by Sutter Instruments (2 mentions)
Micropipette puller, by Sutter Instruments (2 mentions)
Picopump pv820, by World Precision Instruments (2 mentions)
Ix2 slp, by Olympus (2 mentions)
Ix53 microscope, by Narishige (2 mentions)
Femtojet 2 microinjector, by Eppendorf (2 mentions)
Halocarbon oil 27, by Merck Group (1 mentions)
Strain 9750, by Bloomington Drosophila Stock Center (1 mentions)
Phenol red, by Merck Group (1 mentions)
Plasmid miniprep kit, by Evrogen (1 mentions)
Phenol red dye, by Merck Group (1 mentions)
Glass capillaries, by World Precision Instruments (1 mentions)
Pc 10 micropipette puller, by Narishige (1 mentions)
11 protocols using m 152 micromanipulator
1
Drosophila Embryo Microinjection Protocol
2
Drosophila Embryo Microinjection Protocol
3
Microinjection of DNA into Zebrafish Embryos
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DNA samples were microinjected into Danio rerio embryos at the first cleavage 20 min after fertilization using an M-152 micromanipulator (Narishige, Japan) and an air-pressure injector PicoPump PV820 (World Precision Instruments, United States) under an inverted microscope Olympus IX2-SLP (Olympus, Japan). The samples were injected into the yolk under the formed germinal disc at an angle of 45° to the surface with the embryo to maximize sample delivery into the yolk center.
The capillaries used with the outer diameter of 20 µm were pulled from glass capillaries (BF100-50–10, Sutter Instrument, United States) by a Micropipette puller (Sutter Instrument, United States). A 1 nl sample was injected into an embryo within 2.8 × 100 ms.
Vectors DNA were isolated from transformed Escherichia coli TG1 using a Plasmid Miniprep kit (Evrogen, Russia). DNA concentration was determined by spectrophotometry using the extinction coefficient of 0.02 ml/(µg × cm) for double-stranded DNA27 . The obtained DNA was dissolved in PBS with 0.05% phenol red (Sigma-Aldrich, United Kingdom).
The capillaries used with the outer diameter of 20 µm were pulled from glass capillaries (BF100-50–10, Sutter Instrument, United States) by a Micropipette puller (Sutter Instrument, United States). A 1 nl sample was injected into an embryo within 2.8 × 100 ms.
Vectors DNA were isolated from transformed Escherichia coli TG1 using a Plasmid Miniprep kit (Evrogen, Russia). DNA concentration was determined by spectrophotometry using the extinction coefficient of 0.02 ml/(µg × cm) for double-stranded DNA27 . The obtained DNA was dissolved in PBS with 0.05% phenol red (Sigma-Aldrich, United Kingdom).
4
Microinjection of DNA Samples into Zebrafish Embryos
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DNA samples were dissolved in PBS (Biolot, Moscow, Russia) containing 0.05% Phenol red dye (Sigma-Aldrich, St. Louis, MO, USA). DNA concentration ranged from 0.3 to 30 attomoles/nL. DNA samples were microinjected into zebrafish embryos at the first cleavage 20 min after fertilization using an M-152 micromanipulator (Narishige, Tokyo, Japan) and an air-pressure injector PicoPump PV820 (World Precision Instruments, Sarasota, FL, USA) under an inverted microscope Olympus IX2-SLP (Olympus, Tokyo, Japan). The samples (1 nL) were injected within 0.28 s into the yolk under the formed germinal disc at an angle of 45° to the plate to maximize sample delivery into the yolk center. The capillaries used with an outer diameter of 20 µm were pulled from glass capillaries (BF100-50-10, Sutter Instrument, Novato, CA, USA) by a Micropipette puller (Sutter Instrument, Novato, CA, USA). A total of 6750 eggs were injected.
5
Generating Transgenic C. elegans Lines
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Transgenic lines were generated by following standard microinjection procedure [97 ] using an Olympus Ix53 microscope equipped with 20× and 40× lenses, Narishige M-152 micromanipulator (Narishige, Japan), and Eppendorf Femtojet 2 microinjector (local distributors of Eppendorf products). The F2 progeny that inherited and stably expressed the extrachromosomal transgene were UV irradiated to generate integrated lines. Worms with 100% transmission were selected and outcrossed with the wildtype N2 strain five times. Detailed information on the concentration of plasmids and co-injection markers used is listed in S2 Table .
6
Drosophila Transgenesis via PhiC31 System
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All reporter plasmids were integrated into a unique landing site on the third chromosome using VK00033 strain (26 (link)). PhiC31 was maternally provided using vas-phiC31 strain (27 (link)). Microinjection was performed as previously described (28 (link)). In brief, 0–1 h embryos were collected and dechorionated with bleach. Aligned embryos were dried with silica gel for ∼7 min and covered with FL-100-1000CS silicone oil (Shin-Etsu Silicone). Subsequently, microinjection was performed using FemtoJet (Eppendorf) and DM IL LED inverted microscope (Leica) equipped with M-152 Micromanipulator (Narishige). Injection mixture typically contains ∼500 ng/μl plasmid DNA, 5 mM KCl, 0.1 mM phosphate buffer, pH 6.8. mini-white marker was used for screening.
7
Microinjection in Siberian Sturgeon Prolarvae
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A. baerii prolarvae were anesthetized with using 200 mg/L concentration of MS-222 and placed on a 1% agarose bed. Microinjection needles of 20 μm diameter were prepared from glass capillaries (World Precision Instruments, Sarasota, FL, USA) with a PC-10 Micropipette puller (Narishige, Tokyo, Japan) and EG-401 Micro grinder (Narishige). Microinjection was performed manually with a M-152 micromanipulator (Narishige). Injections were made at the border region between the prolarval body and the yolk extension [15 (link)]. The injection was traced using a visible methylene blue staining dye (Sigma-Aldrich) at 0.5 mg/mL. The amount of dye solution injected into each prolarva was estimated to be approximately 15–20 nL depending on injection batches. A group of 7–9 prolarvae was anesthetized for each trial, with eight replicate trials made to yield a dataset of 60 microinjected prolarvae. These trials were carried out at three different age stages (Day 0 to Day 1, Day 2, and Day 3). After injection, prolarvae were transferred to the recovery tank and then to rearing cages for monitoring post-procedure viability for up to 120 h post injection, at 12-h intervals. Two control groups, including an anesthetized but not-injected group, and a non-anesthetized group were also prepared on each day of treatment.
8
Generating Stable Transgenic C. elegans
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Transgenic lines were generated by following standard microinjection procedure (Fire et al., 1998 ) using an Olympus Ix53 microscope equipped with 20× and 40× lenses, Narishige M-152 micromanipulator (Narishige, Japan), and Eppendorf Femtojet 2 microinjector (local distributors of Eppendorf products). The F2 progeny that inherited and stably expressed the extrachromosomal transgene were UV irradiated to generate integrated lines. Worms showing 100% transmission were selected and outcrossed with the wildtype N2 strain five times. Detailed information on the concentration of plasmids and co-injection markers used is listed in Supplementary Table 2 .
9
Efficient Fly Line Generation via Transgenesis
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All the reporter plasmids and NLS-mCherry-MCP expressing plasmid were integrated into a unique landing site on the third chromosome using VK00033 strain59 (link). phiC31 was maternally provided using vas-phiC31 strain60 (link). Microinjection was performed as previously described61 (link). In brief, 0–1 h embryos were collected and dechorionated with bleach. Aligned embryos were dried with silica gel for ~8 min and covered with FL-100-1000CS silicone oil (Shin-Etsu Silicone). Subsequently, microinjection was performed using FemtoJet (Eppendorf) and DM IL LED inverted microscope (Leica) equipped with M-152 Micromanipulator (Narishige). The injection mixture typically contains ~900 ng/μl plasmid DNA, 5 mM KCl, 0.1 mM phosphate buffer, pH 6.8. mini-white marker was used for screening.
10
Endogenous Locus Tagging and Enhancer Deletion in Drosophila
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MS2/PP7-tagging of endogenous locus by CRISPR/Cas9 pCFD3 gRNA expression plasmid and pBS-MS2-dsRed or pBS-PP7-dsRed donor plasmid were co-injected using nanos-Cas9 strains 63 . Microinjection was performed as previously described 64 . In brief, 0-1 h embryos were collected and dechorionated with bleach. Aligned embryos were dried with silica gel for 7 min and covered with FL-100-1000CS silicone oil (Shin-Etsu Silicone). Subsequently, microinjection was performed using FemtoJet (Eppendorf) and DM IL LED inverted microscope (Leica) equipped with M-152 Micromanipulator (Narishige). Injection mixture typically contains 500 ng/ml pCFD3 gRNA expression plasmid, 500 ng/ml pBS-MS2/PP7-dsRed donor plasmid, 5 mM KCl, 0.1 mM phosphate buffer, pH 6.8. 3xP3-dsRed marker was used for screening.
Enhancer deletion by CRISPR/Cas9 pCFD3 gRNA expression plasmids, pBS-3xP3-GFP donor plasmid and pBS-hsp70-Cas9 plasmid (addgene #46294) were co-injected to homozygous hb-MS2 embryos. Microinjection was performed as described in previous section. Injection mixture contains 500 ng/ml pCFD3 gRNA expression plasmids, 500 ng/ml pBS-3xP3-GFP donor plasmid, 500 ng/ml pBS-hsp70-Cas9 plasmid, 5 mM KCl, 0.1 mM phosphate buffer, pH 6.8. 3xP3-GFP marker was used for subsequent screening. Deletion was confirmed by PCR analysis of genomic DNA purified from the resulting mutant.
Enhancer deletion by CRISPR/Cas9 pCFD3 gRNA expression plasmids, pBS-3xP3-GFP donor plasmid and pBS-hsp70-Cas9 plasmid (addgene #46294) were co-injected to homozygous hb-MS2 embryos. Microinjection was performed as described in previous section. Injection mixture contains 500 ng/ml pCFD3 gRNA expression plasmids, 500 ng/ml pBS-3xP3-GFP donor plasmid, 500 ng/ml pBS-hsp70-Cas9 plasmid, 5 mM KCl, 0.1 mM phosphate buffer, pH 6.8. 3xP3-GFP marker was used for subsequent screening. Deletion was confirmed by PCR analysis of genomic DNA purified from the resulting mutant.
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