Sp6 mmessenger mmachine kit

Plasmids (pCS2-H2B-mRFP, pCS2-mib-GFP, pCS2-par-3-GFP, and pCS2-GFP-centrin) were linearized by the restriction enzyme Not I digestion. Not I–linearized plasmids were purified (QIAquick Gel Extraction Kit), and the 5′-capped mRNAs were synthesized using the SP6 mMessenger mMachine Kit (Ambion). For GFP-centrin, H2B-mRFP, mib-GFP, and par-3-GFP mRNA mRNA injection, 4-nl mRNAs at 0.2 to 0.5 μg/μl were mixed with an equal volume of injection buffer containing 0.05% phenyl red and injected into the yolk of a one- to four-cell stage embryos. For par-3-GFP mRNA injection, the mRNAs were injected into single cells at the 32- to 64-cell stages to obtain mosaic expression. All injections were done with an injector (WPI PV830 Pneumatic Pico Pump) and a micromanipulator (Narishige, Tokyo, Japan).