Live Imaging of Early Crozier Embryos

We synthesized mRNAs for microinjections with Ambion’s SP6 mMESSAGEmMACHINE kit. The capped mRNAs produced were diluted in nuclease-free water and used for microinjections in order to detect fluorescence signal in early M. crozieri embryos. Nuclei were marked and followed using histone H2A-mCherry (H2A-mCh) and GFP-Histone (H2B-GFP). The plasmids carrying the nuclear marker pCS2-H2B-GFP (GFP-Histone) and pDestTol2pA2-H2A-mCherry [37 (link)] were transformed, purified and concentrated as described before and then linearized with the restriction enzymes NotI and BglII, respectively. To follow live microtubules, we used a GFP fusion of the microtubule binding domain of ensconsin (EMTB-3XGFP). These clones were the gift of the Bement Lab (University of Wisconsin) [8 (link), 56 (link)] and were commercially ordered from http://addgene.org (EMTB-3XGFP: https://www.addgene.org/26741/).