Generation and expression characterization of LRRK2 KO mice. (a) Schematic diagram (courtesy of Ozgene PLC) showing targeted locus. Exon 41 was flanked with LoxP sites to allow deletion with Cre recombinase. PKG-Neo-pA-SD-IS is Ozgene’s standard selection cassette and was inserted downstream of exon 41. The PKG-neo cassette was also flanked by FRT sites to allow FLPe recombinase deletion. The targeting vector was constructed from three fragments, the 5’homology arm, the 3’ homology arm and the lox P arm, which were generated by PCR. Splicing of exon 40 to 42 causes a frame shift mutation, with the introduction of an early stop codon (TGA). (b) Northern blot hybridized with a probe to LRRK2 exon 24–27 showing absence of transcript in KO and diminished transcript in HET. A histone probe was used as loading control. (c) Immunoblot with LRRK2 antibody 1182E, raised to amino acids 841–960 showing the absence of LRRK2 protein in KO and diminished signal in HET. GAPDH was used as a loading control. (d) Immunohistochemistry with MJFF2 c41-2 antibody showing WT and KO brain sections at the level of the striatum. Specific signal is seen in the WT compared to KO. Rabbit IgG was used as an isotype control. Boxes depict enlarged images to the right. Scale bar 50 microns.