Eight-week-old B6D2F1 females were superovulated with 6 international units of pregnant mare’s serum gonadotropin (PMSG) for 48 h and then injected with 6 international units of human chorionic gonadotropin (hCG), subsequently mated to homozygous Oct4-EGFP males for 12 h. Zygotes were harvested from oviducts of B6D2F1 females with a plug after 24 h post-hCG injection using hyaluronidase (Sigma, Cat# H3884). For zygote microinjection, the mixture of base editor mRNA (20 to 200 ng/μl) and sgRNA (100 ng/μl) was diluted in RNAase-free water, centrifuging at 4 °C, 13,400 g for 10 min, and then injected into the cytoplasm of zygotes in a droplet of HCZB medium containing 5 µg/ml cytochalasin B (CB, Sigma, Cat#C6762) using a micromanipulator (Olympus) and a FemtoJet microinjector (Eppendorf). The injected zygotes were cultured for 24 h to two-cell embryos and 96 h to blastocysts in AA-KSOM (Millipore, Cat#MR-106-D) medium at 37 °C under 5% CO2 in the air. The two-cell embryos were transferred into the oviduct of pseudopregnant ICR females at 0.5 days postcopulation. The blastocysts were used for genotyping and morphological analysis.
Micromanipulator
Manufactured by Olympus
The Micromanipulator is a precision instrument used to precisely control and position microscopic objects or samples. It allows for the fine manipulation of small-scale components with high accuracy and repeatability.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using micromanipulator
1
Zygote Microinjection and Embryo Transfer for Gene Editing
2
Generating Mouse Models Using CRISPR
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Eight-week-old B6D2F1 female mice were superovulated with 6 international units of pregnant mare's serum gonadotropin (PMSG) for 48 h and then injected human chorionic gonadotropin (hCG), subsequently mated to homozygous Oct4-eGFP males for 12 h. Zygotes were harvested from oviducts of B6D2F1 females with plug 24 h post hCG injection using hyaluronidase (Sigma). For microinjection, the mixture of BE3 mRNA (100 ng/μl) and sgRNA (10, 20, 50 and 100 ng/μl) was diluted in RNAase-free water, centrifuged at 4°C, 13 400g for 10 min, and then injected into the cytoplasm of zygotes in a droplet of HCZB medium containing 5 μg/ml cytochalasin B (CB, Sigma) using a micromanipulator (Olympus) and a FemtoJet microinjector (Eppendorf). The injected zygotes were cultured for 16 or 24 h to two-cell embryos in AA-KSOM (Millipore) medium with different concentration of Ricolinostat or Nexturastat A, then cultured in normal AA-KSOM medium for 72 h to blastocyst stage at 37 °C under 5% CO2 in air. Two-cell embryos were transferred into oviduct of pseudopregnant ICR females at 0.5 day post copulation for generating mouse models as previously described (23 (link)).
3
CRISPR mRNA Injection for Transgenic Mice
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The mixture of NG-ABEmax or NG-ABEmax-KR mRNA (100 ng/μl) and sgRNA (100 ng/μl) was diluted in RNase-free water, centrifuging at 4 °C, 13,400 g for 10 min and then injected into the cytoplasm of zygotes harvested from B6D2F1 females (mated with C57BL/6 males) using a micromanipulator (Olympus) and a FemtoJet microinjector (Eppendorf). The injected embryos were cultured in EmbryoMax KSOM Medium (Sigma-Aldrich) for 24 h to the two-cell embryos and 84 h to the blastocysts. Some two-cell embryos were transferred into oviducts of recipients at 0.5 days postcoitum (dpc). Recipient mothers delivered pups at 19.5 dpc and we analyzed phenotypes of offspring at day 10 after birth.
4
Single-cell chemical profiling using InESI-MS
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The cells were incubated with or without 200 μM NCBT in serum-free culture medium for 1 h and maintained in a humid atmosphere of 5% CO2 at 37 °C. Then, the cells were washed with PBS (pH 7.4) three times and finally stored in PBS solution. Single-cell micropipette sampling was performed with a Burleigh micromanipulator and Olympus CK2 inverted microscope. A sampling micropipette containing 185 mM NH4HCO3 and 80 mM NH4Cl was attached to the micromanipulator and then inserted into single cells. The intracellular chemical constituents were obtained from the assayed cells by applying approximately 75 kPa negative pressure through a syringe connected to the glass pipettes for 1 min. Then, the pipette was removed and analysed via the InESI-MS device as previously described.13,23 (link)
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