Wild type and β3GnT2−/− mouse littermates were generated from β3GnT2 het/het crosses. PCR was used to genotype the offspring, with a 700-bp fragment amplified from the wildtype β3GnT2 allele and a 1,071–bp fragment amplified from the β3GnT2−/− allele. β3GnT2−/− mice were established from the KST308 embryonic stem cell line, which harbors a secretory trap vector insertion in the β3GnT2 coding sequence as previously described [8, 37]. Mice were housed according to standard National Institutes of Health and institutional care guidelines, and procedures were approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee (Worcester, MA, USA).
In situ hybridization
Digoxigenin (DIG)-labeled sense and antisense riboprobes were transcribed from cDNAs containing subcloned OR coding sequences using vector-specific RNA polymerases and DIG labeling mix (Roche Molecular Biochemicals, Pleasanton, CA, USA). For in situ analysis of adult mouse OEs, age P28 mice were deeply anesthetized and transcardially perfused with 4% Formaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and further fixed overnight in 4% Formaldehyde at 4°C followed by immersion in 30% sucrose. Bone thickness in the posterior region of the snout from the orbital area to the OB was reduced using surgical knives. The dissected samples were frozen on dry ice, coronally sectioned at 20-μM thickness with a cryostat, and then mounted on Superfrost plus microscope slides, (Thermo Fisher Scientific, Waltham, MA USA). After prehybridization and hybridization, the DIG-labeled RNA hybrids were detected with an anti-DIG Fab fragment conjugated to alkaline phosphatase (Roche Molecular Biochemicals) at a dilution of 1:1,000 overnight at 4°C. The color reaction was produced with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate with levamisole added to block endogenous phosphatase activity.
Histology and Immunocytochemistry
Tissue was prepared by transcardial perfusion using 2% PLP (paraformaldehyde-lysine-periodate) fixation in 0.1 M phosphate buffer (PB), pH 7.4. Antibodies to mOR256-17 are sensitive to fixation conditions and we determined empirically that immunoreactivity on frozen tissue sections was enhanced using 2% PLP compared to 4% paraformaldehyde or other stronger fixatives. Heads were subsequently removed and postfixed 16 hrs in the same fixative solution, followed by cryoprotection in 30% sucrose. After embedding, tissue sections were prepared on a Microm HM505E cryostat at 50 μM thickness and were then immediately thawed in transwell boats filled with PBS for staining as free floating sections. Tissues were blocked for one hour in 2% BSA, then incubated overnight at 4°C with the primary antibody for mOR156-17  or mOR28  and diluted in 1% BSA/PBS/0.3% Triton X-100. After washing, tissue sections were further incubated for two hours at room temperature with species-specific secondary biotinylated antibody and visualized with the Vectastain ABC peroxidase kit and 3,3’-diaminobenzadine tetrahydrochloride (Vector Laboratories, Burlingame, CA USA), or with Cy3-conjugated (Jackson Immunoresearch, West Grove, PA USA) or Alexa Fluor 488 (Invitrogen, Grand Island, NY USA) conjugated secondary antibodies. Images were captured using a Zeiss Axioplan photomicroscope equipped with a Spot RT camera (Diagnostic Instruments (Sterling Heights MI USA).
Specific OR-expressing neurons in PD28 OEs of β3GnT2−/− and WT control mice were quantified using a Nikon Eclipse E400 microscope (MVI, Inc., Avon MA USA) at 100X. The complete OE cavity was serial sectioned at 20 μM and collected on 20 to 22 slides per case. Each in situ hybridization experiment included a pair of β3GnT2−/− and WT slides for each OR probe. Each probe was used on three different pairs of mice. All the sections from one slide of each genotype were counted and used for analysis. These counts were compared using a paired two-way ANOVA to control for any variability, which may occur between each in situ experiment. In addition, pairs of slides within a case, using one probe, were counted and compared and found to be less than 3% different, which was not significant (NS). Thus, we extrapolated the total amount of labeled cells for each OR by adjusting the counts to the total number of slides in each case (Table 2). Statistical analysis was performed using SigmaStat 2.0 (Systat software, San Jose, CA USA).
Real time reverse transcriptase quantitative PCR
RNA was extracted from microdissected olfactory epithelia of 6 β3GnT2+/+ and β3GnT2−/− mice at six months of age using Trizol Reagent (Invitrogen), and cDNA prepared as described previously (Henion et al., 2011). Quantitative PCR (qPCR) was performed with oligonucleotides designed using online Primer3 software (version 0.4.0). Primer sequences used for template amplification are available upon request. Samples were amplified in triplicate using GoTaq Polymerase (Promega, Fitchburg, WI, USA) in a StepOnePlus Real-Time PCR System (Applied Biosystems, Life Technologies, Grand Island, NY, USA). Relative gene expression levels were determined by the Comparative CT (threshold cycle) method after normalization to RNA polymerase 2 as an endogenous reference.
Gene array hybridization
OEs from adult mice were collected in PBS on ice. Tissue samples were homogenized using TRIzol (Invitrogen, Carlsbad, CA, USA) and the RNA was collected in the aqueous phase after chloroform extraction. Subsequently, Isopropyl Alcohol was used to precipitate the RNA, followed by washing with 75% ethanol, air-dried, and redissolved in RNAse-free DEPC H2O. After this RNA isolation, a Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA) was used to further purify the RNA, and the yield analyzed with a DU 650 Spectrophotometer (Beckman Coulter Inc, Brea, CA, USA). Yields per mouse OE measured at an absorbance of 260 nm were 10 to 20 ug RNA.
Affymetrix gene array technology was used to compare the gene profiles between β3GnT2 WT and mutant mouse strains. Individual samples were hybridized to 12 gene chips in a high-density oligonucleotide array (Affymetrix Mouse Gene 1.0 ST Array, 28,853 mouse genes (1,100 OR genes)/chip) that uses 25-mer probes distributed across the transcribed regions of each gene, with a median of 26 probes per gene. The Ambion WT Expression kit (Applied Biosystems, Foster City, CA, USA) was used to synthesize first and second strand cDNA, and generate the purified sense strand for the Affymetrix gene chip WT Terminal labeling kit (Affymetrix, Santa Clara, CA, USA). The single stranded cDNA produced by the Ambion kit was fragmented and labeled using the GeneChip WT Terminal Labeling Kit. The product was then hybridized to the chip for 16 to 17 hours at 45°C, washed, stained and scanned using the Affymetrix GeneChip Array Scanner. The log based signal values were generated using the RMA algorithm in Expression Console. For each pair of samples, results of each probe were compared and an “R” value was calculated. The median of the “R” values of each probe set (R_median) was used for filtering the potentially differential gene expression. No normalization was conducted.
Olfactory discrimination task
β3GnT2+/+ and β3GnT2−/− mice 9 to 13 weeks old were trained in an olfactory discrimination task . Pairs of odors, each known to elicit strong cAMP responses were used in the sand buried food task to measure an animal’s ability to associate an odor with a reward . The mice were food-deprived for 24 hours before the first day of testing began then maintained on a restricted diet throughout the four day trial. On Day 1, the mice were allowed to locate a food reward in the sand. Day 2 was a training day in which each mouse was presented with a food reward in the sand, which was associated with a test odor. The test odors were shifted to different positions within the cage during the training and subsequent testing days. The training period consisted of six 20-minute time periods with 10-minute intervals, where each mouse would have an opportunity to locate the food reward. Early in the training the mice were allowed additional time, if necessary, to locate the food reward. On Day 3, mice were given a choice between a novel odor and the same test odor presented on Day 2. Subsequent sets of six trials each on Days 3 and 4 were scored either correct or incorrect accordingly to which dish each mouse would begin digging. Days 3 and 4 consisted of two separate sets of trials. In the first set of trials the mice were tested without a buried food reward, while in the second set of trials the reward was present.