One-cell stage wildtype AB or Smoc1ug104; BRE:GFP; H2B-mCherry embryos were injected using standard procedures with different smoc1 mRNA concentrations. This was generated by linearization of pCS2-smoc1 vector with NotI (NEB), and transcribed using the SP6 mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit (Ambion). The same procedure was performed to obtain smoc1-mCherry, smoc1ug104-mCherry and Cas9 mRNAs. For antisense morpholino oligonucleotide (MO) injections, 2ng or 12ng of a splicing block MO (5′-CCGGAACTCTGACAGACCTGAGCAA-3′) (Abouzeid et al., 2011 (link)) and its respective mismatch control MO (5′-CCGCAAGTCTCACACACCTCAGCAA-3′) were used (Gene Tools, LLC) in BRE:GFP; H2B-mCherry heterozygote embryos. Embryos were left to develop at 28.5°C until the desired stage and then processed for SEM, live imaging or immunostainings. A PV-820 Pico-injector (World Precision Instruments) and a Narashige micromanipulator were used for microinjection.
Pv 820 pico injector
Manufactured by World Precision Instruments
Sourced in United States
The PV-820 Pico-injector is a precision microinjection system designed for the accurate and controlled delivery of small volumes of fluids. It features a high-resolution stepping motor for precise control of injection volumes down to the picoliter range.
Automatically generated - may contain errors
2 protocols using pv 820 pico injector
1
Spatiotemporal Regulation of Smoc1 in Zebrafish Embryos
2
Evaluating Au-nanobeacon silencing efficacy
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The fli-EGFP transgenic zebrafish line was chosen due to its EGFP stained vasculature, providing an easy model to evaluate the silencing efficacy of the Au-nanobeacon. Post-fertilized fli-EGFP embryos were collect and embryos at the sphere stage were divided in different groups and injected with: 50 nM of AuNPs@citrate and 50 nM of AuNPs@PEG as controls and 150 nM of Au-nanobeacon, using a PV-820 Pico-injector (World Precision Instruments, Sarasota, FL, USA) and a Narashige micromanipulator. The free oligonucleotides molecules were also injected at the same concentration administered in the Au-nanobeacon. The embryos were incubated in 90 cm Petri dishes (Sarstedt, Germany) at 28 °C using embryo medium, until 24 h post-fertilization, a time-point where blood vessels are already formed and therefore the levels of EGFP expression could be evaluated.
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