Generation of Rab39b knockout mice
Mice were housed in a specific-pathogen free environment under a 12 hour light/dark cycle with free access to food and water. Rab39b knockout mice were generated using CRIPSR/Cas9 by the South Australian Genome Editing Facility (Adelaide, Australia). In brief, CRISPR guides flanking exon one of Rab39b (supplementary Table 1) were injected into c57Bl/6J mouse zygotes, and the resulting genotypes confirmed by Sanger sequencing of genomic DNA extracted from tail biopsies (supplementary Table 2). Potential Cas9 off-target sites were predicted using the CRISPR/Cas9 target online predictor (CCTop). Colonies were established by backcross of hemizygous KO male mice to the wildtype females for a minimum of five generations prior to tissue collection and data analysis.
RAB39B recombinant protein production, purification and antibody development
E. coli BL21(RIL) pRARE competent cells (Invitrogen) were transformed with pET28a-GST_RAB39B and grown at 30°C to an OD600 of 0.5. Recombinant protein expression was induced with 0.5 mM IPTG for four hours and GST-tagged proteins were purified using the GST-BindTM Kit (Novagen). For generation of mouse monoclonal antibodies, 100 µg of purified GST-RAB39B and 200 µg of poly (I/C) adjuvant were injected intraperitoneally into two months old BALB/c female mice. Three injections were performed at two week intervals. Spleen cells were fused with Sp2/0.Agl4 myeloma cells and hybridoma culture supernatants were tested by ELISA for reaction with GST-RAB39B. Specific cultures were cloned twice on soft agar. Two specific clones, 1H1 and 1B8, were established and ascites fluid was prepared by injection of 2 × 106 hybridoma cells into Freund adjuvant-primed BALB/c mice. All animal experimental procedures were performed according to the European authority guidelines.
RNA extraction and PCR
Total RNA was extracted from brain tissue using the SV Total RNA Isolation System (Promega) according to the manufacturer’s protocol and RNA integrity was evaluated using Nanodrop ND-2000 (Thermo Scientific). cDNA was synthesized from one mg of total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche). PCR analysis of cDNA was performed using MyTaq DNA Polymerase (Bioline) with a T100 Thermal Cycler (Biorad) (supplementary Table 2). Amplified products were resolved by gel electrophoresis in 1% Agarose (Bioline). Quantitative real time PCR (qRT-PCR) analysis was performed with a LightCycler LC480 II (Roche) and taqman probes (Life Technologies) specific for mouse Rab39b (Mm00838519_m1) and Tbp (Mm00446971_m1). Relative expression was calculated using the Pfaffl ddCT method(10).
Protein extraction and western blotting
Western blot analysis was performed as previously described (11). Briefly, total protein was extracted from brain tissue using a buffer containing 2% SDS and 1x Protease inhibitor (Sigma). Lysates were sonicated and total protein concentration estimated by Bicinchoninic acid assay (Pierce). For micro-dissections, whole brains were sliced into 2 mm coronal sections using a mouse brain slicer matrix (Zivic), then further dissected to isolate specific brain regions under a stereomicroscope (Leica). Western blot analysis was performed using 20 µg of total protein separated on 12% SDS-PAGE and transferred onto 0.45 µm-pore PVDF membranes (Immobilon-P) at 10V overnight. Following transfer, membranes were blocked in 5% skim milk for two hours, then incubated with primary antibodies at 4°C overnight. Antibody binding was revealed using horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratories) and enhanced chemiluminescence (Bio-Rad) according to manufacturer’s protocol. Images were captured with an ImageQuant LAS4000 and quantified using ImageQuantTL software (GE Healthcare).
In-situ hybridization (ISH)
ISH was performed essentially as previously described (12). Briefly, mice were culled by cervical dislocation and whole brains were immediately dissected, fixed in 4% paraformaldehyde (PFA) overnight, cryoprotected in 20% sucrose overnight at 4°C, and then snap frozen in iso-pentane over dry-ice. Serial sections of 10 µm thickness were cut and collected on superfrost adhesion slides (Menzel-Glaser). To generate sense and anti-sense ssRNA riboprobes, the target Rab39b exonic sequences were amplified using specific primers and cloned into PCRII TOPO plasmid (Invitrogen) (supplementary Table 3). Riboprobes were Digoxigenin (DIG) labelled using the DIG RNA labelling kit (Roche) according to manufacturer’s instructions and hybridized to tissue sections at 64°C in a sealed chamber overnight. Probe binding was detected by incubation with an alkaline phosphatase conjugated anti-DIG antibody (Roche, 11093274910) and visualized through NBT/BCIP reaction (Roche). Slides were mounted with aqueous mounting agent (Merck) and viewed and imaged with an optical microscope (Olympus).
Immunohistochemistry (IHC)
Mice were culled by cervical dislocation and whole brains were immediately dissected, fixed in 4% PFA overnight, then cryoprotected in 30% sucrose for up to one week at 4°C before sectioning. Serial sections of 10 µm thickness were collected on superfrost adhesion slides (Menzel-Glaser). Slides were blocked and permeabilized in 0.2% TritonTM X-100 (Sigma), 3% Bovine serum albumin (Sigma) and 10% Goat serum (Life Technologies) for one hour, incubated with primary antibodies for 48 hours at 4°C, then secondary antibodies (AlexaFluor 488 or 594, Invitrogen) for one hour. Slides were mounted with Vectashield containing DAPI (VectorLabs) and analyzed with a confocal microscope (LSM 780, Carl-Zeiss) using ZEN black image processing software. Primary antibodies utilized include rabbit anti-RAB39B (Proteintech, 12162-1-AP), sheep anti-tyrosine hydroxylase (TH) (Merck Millipore, ab1542), and mouse anti-microtubule-associated protein 2 (MAP2) (Sigma Aldrich, M1406).
Statistical analysis
Statistical significance was determined using unpaired student t-tests (Graphpad Prism 7, CA, USA). All quantified data are displayed as mean +/− standard error of the mean (SEM).