Traditional western blotting was performed as described previously14. mice, indicating that extra Reelin signaling is usually detrimental to hippocampal layer formation. The neuronal dendrites of PA-DV KI mice experienced more branches and were elongated compared to wild-type mice. These results present the first direct evidence of the physiological importance of Reelin cleavage. mice (B6C3Fe-a/a-rl) were purchased from Jackson Laboratories and back-crossed with Jcl:ICR mice. A knock-in mouse strain in which Pro1,244 and Ala1,245 of Reelin were substituted with Asp and Val residues (PA-DV KI mice) was generated as follows: Mutations were introduced into the mouse Reelin gene using the CRISPR/Cas9 system. A linker corresponding to the sgRNA sequence was generated using primers CACCGTTCATAGGGTAATCGAAAGC and AAACGCTTTCGATTACCCTATGAAC and inserted into the Bbs1 Imidazoleacetic acid site of the px330 plasmid56. This plasmid and synthetic single-strand DNA (GTTATCCAACCGTCTTCCTGTTTCGTTTGTGTTGTCCCTAATAGAACTTCTATGAGAAGGACGCTTTCGATTACCCTATGAACCAAATGAGTGTGTGGCTAATGTTGGCCAATGAAGGC) were injected into the fertilized egg pronucleus of C57BL/6?J mice. Genomic DNA was prepared as explained previously14 and genotype was determined by Rabbit Polyclonal to EFEMP1 PCR (33 cycles at 94?C for 30?s, 64?C for 30?s, and 72?C for 30?s) using the following primers: for WT allele, PDKI-WT-21 (CCCTAATAGAACTTCTATGAGAAGCCA) and PDKI-Rev-24 (CCTTACCAATAGAGCTGAAAGGCTTC); for PA-DV KI allele, PDKI-WT-21 and PADV-KI-93 (CCCTAATAGAACTTCTATGA GAAGGACGT). The sizes of the PCR products were 347?bp and 349?bp for WT and PA-DV KI alleles, respectively. The PA-DV KI mice were back-crossed with either C57BL/6?N or with Jcl:ICR at least 8 occasions. All experiments shown were performed with C57BL/6?N background, except for Fig.?4. In Fig.?4, the mice with Jcl:ICR mice were analyzed. Immunohistochemistry Immunohistochemistry was performed as explained previously12,14. All data shown in the Figures are representative images of at least three impartial mice, otherwise indicated. GolgiCCox staining GolgiCCox staining was performed as explained previously12. Briefly, dissected P14 brains were Imidazoleacetic acid immersed in the GolgiCCox answer for three days, followed by seven days in PBS made up of 30% sucrose. Brains were slice at a thickness of 160 m sections using a VT1000S vibratome (Leica Microsystems). Sections were incubated in 15% aqueous ammonium hydroxide for 30?min under a fume hood in the dark followed by 30?min?in Kodafix answer (CosmoBio). Sections were rinsed with distilled water twice, and dehydrated using an ethanol series and lemosol and mounted with Softmount (Sakura). Layer V neurons which located in the somatosensory area using a light microscope with a 60 lens, and pictures were taken with the BZ-9000 system (Keyence). The primary and secondary dendrite lengths were measured using ImageJ with the NeuronJ plugin (National Institutes of Health) as explained previously12. The length of secondary dendrites that branched from the primary dendrite within 80 m of the soma were counted and measured. Experts were blinded to the genotypes of the mice in all experiments. Cell culture and transfection The culturing of human embryonic kidney (HEK) 293?T cells, transfection of plasmid DNA using Lipofectamine2000, and recovery and preservation of the culture supernatants were performed as described previously57C59. Western blotting Western blotting was performed as explained previously12,14. Dissected cerebral cortices, hippocampi, and cerebellums were homogenized in lysis buffer (20?mM Tris-HCl, pH 7.5, 150?mM NaCl, 5?mM EDTA, 1% Triton X-100, 0.1% H2O2, and 5?mM Na3VO4)57. Insoluble debris was removed by centrifugation (10?min; 13,000?rpm), and the supernatants were collected. Western blotting was performed as explained previously14. Imidazoleacetic acid The blots were analyzed with ImageJ and quantified as explained previously59. All natural images are offered in Supplementary Figures which include all uncropped initial data that were used to prepare Figs. ?Figs.1,1, ?,22 and ?and44. Statistical analyses Data are offered as the mean the standard error of the mean (SEM). One-way ANOVA followed by the Tukey-Kramer ad hoc test was used to compare three different groups. Two-tailed Students test were used to compare the means of two groups. Statistical analyses were performed with Prism6 (GraphPad Software). Statistical significance was represented as *p? ?0.05, **p? ?0.01, ***p? ?0.001, and ****p? ?0.0001. Supplementary Information Supplementary Information.(26M, pdf) Acknowledgements We thank Drs. Tom Curran (Childrens Research Institute at Childrens Mercy Hospital, MO, USA), Junichi Takagi (Osaka University or college, Japan), and Katsuhiko Mikoshiba (Brain Science Institute, RIKEN, Japan) for generously providing reagents. This work was supported by JSPS KAKENHI (17H03895 and 17K19500 to M.H., 17K08281 Imidazoleacetic acid to T.K., and 17J10967 to H.Ogino), ACT-M (16im0210602h0001 and 17im0210602h0002) of the Japan Agency for Medical Research and Development (to M.H.), and Ono Medical Research Foundation (to M.H.). H. Ogino is usually Research Fellow of Japan Society for the Promotion of Science (DC1). Author contributions E.O., H.Ogino., T.K., and M.H. conceived and designed research; E.O., H.Oishi., Imidazoleacetic acid T.K. and M.H. established the PA-DV KI mice.; E.O., H.Ogino., T.S. and Y.Y. performed biochemical and molecular biological experiments; E.O., H.Ogino., T.S., Y.Y. and H.T. performed immunohistochemical experiments; E.O., H.Ogino., T.S., Y.Y. and M.H. performed statistical analyses; E.O., H.Ogino., T.K. and M.H. published the manuscript. Competing interests This.