Platynereis zygotes were injected using a Zeiss Axiovert 40C inverted microscope, equipped with a joystick micromanipulator (Narishige) and a microinjector (FemtoJet, Eppendorf). Injection needles were pooled from glass capillaries (borosilicate thin wall with filament, 1.0 mm outer diameter, 0.78 mm inner diameter, Harvard apparatus) using a Sutter needle pooler. Embryos were accommodated on an agarose stage, customized for Platynereis injections.
GCaMP6s (Chen et al., 2013 (link)) was kindly provided by the GENIE Project, Janelia Farm Research Campus, Howard Hughes Medical Institute (Addgene plasmid 40753). The GCaMP6s coding sequence was subcloned in the pCS2+ vector, and mRNA was in vitro transcribed with the SP6 mMessage mMachine High Yield Capped RNA Transcription Kit (Ambion). GCaMP6s mRNA was injected in the CD blastomere to a final concentration of 250 μg/μl. H2B-RFP mRNA was co-injected at the same concentration in order to label the CD lineage.
GCaMP6s (Chen et al., 2013 (link)) was kindly provided by the GENIE Project, Janelia Farm Research Campus, Howard Hughes Medical Institute (Addgene plasmid 40753). The GCaMP6s coding sequence was subcloned in the pCS2+ vector, and mRNA was in vitro transcribed with the SP6 mMessage mMachine High Yield Capped RNA Transcription Kit (Ambion). GCaMP6s mRNA was injected in the CD blastomere to a final concentration of 250 μg/μl. H2B-RFP mRNA was co-injected at the same concentration in order to label the CD lineage.
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