We followed institutional guidelines for animal care and research protocols, and approved by the Etiska Nämnden on animal use (ethical permit N241/14). Xenopus laevis eggs were obtained by injecting frogs with 700 units of human chorionic gonadotropin (Pregnyl®, Merck Sharp & Dohme). The embryos were fertilized using a sperm suspension and were dejellied with 1% thioglycolic acid at two-cell stage, and cultured in 0.2× Marc’s Modified Ringer’s solution (MMR) at 18-21 °C. Staging was according to Nieuwkoop and Faber [31 ]. Microinjections were performed in 4% Ficoll/0.3× MMR. The maximum injection volume was 20nl per embryo. The embryos were then cultured in 0.2X MMR until either stage 10.25 (for TOPFlash assay) or stage 32 (for the PCP phenotypes). The mMessage mMachine® sp6 Kit (Ambion) was used to synthesize in vitro capped mRNA. Lrrk2 construct used for the experiments was pCS2-5Xmyc-Lrrk2 [32 (link)]. pCS2-super was generated by inserting an oligonucleotide fragment containing a polylinker sequence (EcoRI, PacI, SbfI, XmaI, XhoI, AscI, XbaI) into the EcoRI/XbaI sites of pCS2. To generate pCS2-beta-galactosidase, beta-galactosidase with nuclear localization signal was obtained by RT-PCR from pBSApBpACAGftILn [33 (link)], and subcloned into the PacI/AscI sites of pCS2-super.
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