Mouse Lines
The following mouse lines were used in our studies:
• Omp-lacZ knockout allele mouse line (Mombaerts et al., 1996)
-GFP (MOR23-IRES-Tau:GFP) allele knock-in mouse line (Vassalli et al., 2002)
53
• M72 -Cre (M71-IRES-Cre) allele knock-in mouse line (Li et al., 2004)
54
• V1rb2
-lacZ (M72-IRES-Tau:lacZ) allele knock-in mouse line (Feinstein and Mombaerts, 2004)
55
• Adcy3 knockout allele mouse line (Wong et al., 2000)
-lacZ (V1rb2-IRES-Tau:lacZ) allele knock-in mouse line (Rodriguez et al., 1999)
• Nrp1 conditional null allele mouse line (Gu et al., 2003)
• Rosa26-lacZ (Rosa26promoter-Stop56
• Rosa26-RFP (Rosa26promoter-Stop56-tdRFP) transgenic mouse line (Luche et al., 2007)
-lacZ) transgenic mouse line (Soriano, 1999)
In study 2, we generated TrpC2-IRES-Cre-IRES-GFP transgenic mice lines (see further).
Mice were bred on a C57BL/6 background. Animals were housed and handled in accordance with the guidelines and regulations of the institution and of the state of Geneva, Switzerland. The day of birth was considered as PND1.
Cloning Strategy for TrpC2-IRES-Cre-IRES-GFP
We modified a BAC (BAC RP23-3D10) that contained the TrpC2 gene. We used homologous recombination in bacteria (Lee et al., 2001; Yu et al., 2000) to modify the 3’ UTR of TrpC2. The targeting vector was based on a
56 The Stop cassette consists of a PGK-Neo-4x pA sequence, flanked by loxP sites.
pBluescript 2 KS(-) backbone with an IRES-Cre-IRES-GFP-frt-Puror-Zeor-frt cassette flanked by homology arms corresponding to the sequences 5’ and 3’ of TrpC2 stop codon, respectively (Figure 38). The primers used to generate homology arms were57:
• 5’ arm, fwd: 5’-gctAGCTCCCGCTCCTGGAGA-3’
• 5’ arm, rev: 5’-ttaattaaTTA
• 3’ arm, fwd: 5’-gagctcGGCAACAGAAGGGTCTCTTCTGTCA-3’ GGACTCGCCCTTGGTCTCCAGAT-3’
• 3’ arm, rev: 5’-ggtacctcgAGCTCCCAGTCTTCCAGTCTCCTA-3’
The 5’ homologous arm is 124bp long and ends with TAA (stop codon of TrpC2).
The 3’ homologous arm is 350bp long, the first 145bp corresponding to the untranslated part of the last exon.
Figure 38: Cloning Strategy to generate the TrpC2 BAC transgenic mouse line. Just after the TrpC2 stop codon, we introduced by homologous recombination in a BAC, an IRES-Cre-IRES-GFP cassette, followed by puromycin (P) and zeocin (Z) resistance genes flanked by frt sites (white diamonds). After homologous recombination, the selection cassette was removed (by inducible expression of flippase in bacteria). The question mark in the first exon of TrpC2 indicates the unknown nature of the 5’ end of the olfactory TrpC2 transcript (multiple splice variants are known). CDS: coding sequence, GFP, green fluorescent protein, i: internal ribosome entry site sequence, P: puromycin resistance, Stop: TrpC2 stop codon (TAA), TV: targeting vector, Z: zeocin resistance.
The BAC was first electroporated in EL250 E.coli strain (Lee et al., 2001). This strain carries a defective prophage containing the λ genes from cI857 to int. The gam and red genes, exo and bet, are under the control of PL (left promoter), which is repressed by the temperature-sensitive repressor cI857 at 32°C and derepressed at 42°C. Gam inhibits the RecBCD nuclease from attacking linear DNA, and Exo and Beta generate recombination activity for that linear DNA. The araC–PBADflpe cassette replaces the segment from cro to bioA. The promoter of the araBAD operon (PBAD), which can be induced by L-arabinose, controls the expression of the flpe gene58. This strain has to be grown at temperature lower than 32°C. Growing at 37°C can partially derepress λ genes and cause genomic or BAC rearrangements, which are very difficult to detect.
57 Lower cases indicate bases added to generate restriction sites needed for subsequent cloning, underscores indicate TrpC2 stop codon reverse complement.
58 Coding for enhanced flippase recombinase. This enzyme is able to recognize and recombine two frt sites (similar to loxP sites recognition and recombination by Cre recombinase).
Homologous recombination was induced by shifting EL250 bacteria containing RP23-3D10 to 42°C prior to electroporation with the linearized targeting vector.
Growing the cells in LB containing 0.1% arabinose induced the removal of the selection cassette. We selected clones that underwent both recombination events correctly, extracted and purified BAC DNA from one of them and injected it in B6D2F1 embryo pronuclei. Founders (screened by PCR) were then bred to C57BL/6 mice to evaluate germline transmission.
Whole Mount Analyses
Animals were dissected, the heads fixed by immersion in 4% paraformaldehyde at 4°C for 5 minutes and stained with X-Gal as previously described (Rodriguez et al., 1999). Whole mount images were taken on a Leica MZFLIII or Zeiss SteREO Lumar.V12 fluorescence-equipped binoculars.
Microscopy
Wide field images were taken on a Zeiss Axioplan2 wide field microscope.
Confocal images were taken on a Leica SP2 or on a Zeiss 510 Meta confocal microscope. All sections were counterstained with DAPI (5 minutes, 1μg/ml) and slides were mounted using DABCO anti-fade mounting medium (Sigma).
RT-PCRs
Tissues were harvested and immediately processed using the Qiagen RNeasy Mini Kit. The following primers were used to amplify Adcy3 transcripts:
• Fwd: 5’-ATCCCAAATTCCGGGTCATCAC-3’
• Rev: 5’-GGAAGCCGTACTCTCGAAGGAT-3’
Amplifications were performed under the following conditions: 5 minutes at 95°C, followed by 34 cycles of 1 minute at 95°C, 1 minute at 60°C, and 2 minutes at 72°C, and a final extension of 10 minutes at 72°C.
In Situ Hybridizations
Tissues were embedded in OCT without fixation, and 14μm cryostat slices were mounted on Superfrost+ slides (Menzel-Glaser), dried for 40 minutes, fixed for 20 minutes at 4°C with 4% paraformaldehyde and hybridized overnight at 65°C in the following buffer: 1x salt buffer (10mM NaCl, 5mM NaH2PO4·H2O, 5mM Na2HPO4·2H2O, 5mM EDTA, pH 7.5), 50% formamide, 10% dextran sulphate, 1μg/μl tRNA, 1x Denhardt’s, with 20ng/μl cRNA probe(s). The Adcy3 probe
contained a 604bp fragment starting 60bp before the start codon. For M72 (M72-lacZ) and P2 (P2-lacZ), or MOR23 (MOR23-GFP) and Omp (Omp-GFP) transcript identification, probes containing a 1.8kb HindIII fragment of lacZ or the entire GFP coding sequence were used, respectively. The Mor28 (olfr1507) probe contained a 404bp fragment corresponding to the 5’UTR (ranging from position -4578 to -4175 before start codon). The olfr727 probe contained the entire coding sequence (975bp). Following hybridizations, slides were washed twice at 65°C and once at RT with 1x SSC, 50% formamide and 0.1% Tween 20.
Slides were pre-incubated in 1x MABT, 2% blocking reagent (Roche) for 30 minutes followed by a 1-hour incubation with alkaline phosphatase-anti-digoxigenin antibody (1:1000, Roche) or/and peroxidase-anti-fluorescein antibody (1:200, Roche). Peroxidase activity was revealed by washing slides three times with TNT (150mM Tris, 150mM NaCl, 0.05% Tween 20, pH 7.5), incubation for 30 minutes with a biotinyl-tyramide solution (PerkinElmer, 1:50), three washes with TNT and incubation for 30 minutes with streptavidin-Alexa 488 (Invitrogen, 1:100). Alkaline phosphatase activity was detected by incubating slides with Fast Red substrate (DAKO) for 30 minutes. Sections were mounted in DABCO mounting medium (Sigma). Fluorescein and digoxigenin-labeled RNA probes were prepared using the DIG RNA Labeling Kit (Roche) following the manufacturer’s instructions.
Affymetrix Oligonucleotide Microarray Hybridization
Main olfactory epithelium RNA from three control and three Adcy3-/- PND26 mice was independently extracted (generating two sets of RNA triplica). These RNA samples were used to generate biotinylated cRNA, which was subsequently hybridized to Affymetrix Mouse Genome 430 2.0 arrays (according to the Affymetrix protocol). To identify differentially expressed transcripts, pairwise comparisons between samples from control and mutant animals were carried out. Genes for which the concordance in the pairwise comparisons exceeded the threshold of 77% (seven out of nine comparisons) were considered to be statistically significant. A concordance of nine indicates that all samples from one condition (control vs knockout) gave signals significantly superior to each sample of the other condition.
Western Blots
Tissues were homogenized in 10mM Tris-HCl (pH 7.6), 1mM EDTA, 100mM NaCl, 100μg/ml PMSF, 1μg/ml aprotinin. Protein extracts (15μg) were loaded on 10% Bis-Tris NuPAGE gels (Invitrogen), and transferred to PVDF membranes.
Membranes were incubated for 16 hours with an anti-AC3 antibody (1:500, sc-588 Santa Cruz Biotechnology), followed by incubation with an alkaline phosphatase-conjugated anti-rabbit antibody. The phosphatase was revealed with the CDP-Star chemiluminescent substrate (WesternBreeze, Invitrogen).
Immunohistochemistry
Tissues were fixed for 2 hours (AC3 labeling) or overnight (for all other antibodies), in 4% paraformaldehyde at 4°C, and embedded in OCT. Cryostat slices (10-14μm) were mounted on Superfrost+ slides. When necessary, post-sectioning acetone fixation (anti-IP3R-3, 5’ at -20°C) or citrate buffer antigen retrieval (anti-carbonic anhydrase type 2, 20’ in 85°C citrate buffer) was performed. Slides were pre-incubated in 0.5% Triton, 10% FCS (except for anti-AC3: 0.1% FCS), 1x PBS for 30 minutes at room temperature. Goat anti-rodent-OMP (1:200-800, Wako Laboratory Chemicals), goat anti-rat-Neuropilin (1:100, AF566, R&D Systems), goat anti-mouse-carbonic anhydrase type 2 (1:400, CAII (M-14): sc-17244, Santa Cruz Biotechnology), goat anti-β-galactosidase (1:400, ab12081, Abcam), rabbit anti-mouse-AC3 (1:400, sc-588, Santa Cruz Biotechnology), rabbit anti-rTrp2 (1:400, (Liman et al., 1999)), rabbit anti-GFP (1:1000, ab290, Abcam), rabbit anti-DsRed(RFP) (1:500, ab36771/ab34771, Abcam), chicken anti-GFP (1:1000, ab13970, Abcam), mouse anti-rat-NCAM (1:500, Sigma) and mouse anti-human-IP3R-3 (1:400, 610312, BD Biosciences) antibodies were used. After overnight incubation at 4°C, slides were washed three times with 0.5% Triton, 1x PBS for 10 minutes and stained with DAPI for 5 minutes (1μg/ml). Primary antibodies were revealed with FITC-conjugated goat anti-chicken (1:400, Abcam), FITC-conjugated goat anti-mouse (1:400, Abcam), Alexa 488-conjugated donkey anti-goat (1:200-800, Invitrogen), Alexa 488-conjugated chicken anti-rabbit (1:400, Invitrogen), Cy3-conjugated donkey anti-rabbit (1:250, Jackson Laboratories), Cy3-conjugated goat anti-rabbit (1:400, Invitrogen), Alexa 555-conjugated donkey anti-mouse (1:500, Invitrogen) or Alexa 555-conjugated donkey anti-goat (1:800, Invitrogen) secondary antibodies. Slides were mounted with DABCO antifade reagent (Sigma). To evaluate the specificity of the anti-AC3 antibody, the peptide used to raise the antibody (Santa Cruz) was pre-incubated for 2 hours at room temperature with the AC3 antibody [10x excess (w/w)].
Appendix
Supplementary Figures
Figure 39: Downregulated transcripts in the main olfactory epithelium of Adcy3 knockout mice.
The most downregulated genes are listed first. Genes with a change fold average inferior to 2 are not listed. A NPM value of 3 indicates that the gene was considered present in each triplicate. The change fold average is considered reliable if both NPMs are at 3 and the pValue is under 0.05
Figure 40: Upregulated transcripts in the main olfactory epithelium of Adcy3 knockout mice. The most upregulated genes are listed first. Genes with a change fold average inferior to 2 are not listed. A NPM value of 3 indicates that the gene was considered present in each triplicate. The change fold average is considered reliable if both NPMs are at 3 and the pValue is under 0.05
Acknowledgements
Obviously, I would first like to thank Professor Ivan Rodriguez for hosting me in his lab. From my first day as a PhD student, he taught me a lot about life science and maybe even more about life. I really appreciate his calm temper, especially when I lose mine, and his sense of humor that helps relieving the pressure. I was lucky to be able to interact directly with him all the time, a situation that is priceless to me.
I want to thank Dr. Daniele Roppolo from the bottom of my heart for all he did when he was around in the lab, all supporting chats and the good advices (about science, about life, about pizza…). I really wish I could see him more often.
I would also like to thank both Professor Denis Duboule and Professor Paul Feinstein for evaluating my work and accepting to be members of my thesis jury.
Of course, I want to say a big “thank you” to all the past and present members of the “IRlab” for their advices, good mood, sense of humor, kindness… and for putting up with me… (Sorry about that, guys)
I am truly thankful to Dr. Luca Capello for all the time he spent with me, sharing his knowledge, making me discover a bit of the GNU/Linux world and helping me out. I wish I could give him a little back sometime, but I still do not know how…
Thanks a lot as well to people from the Galliot Lab and from the Duboule lab (the neighbors) as well as other members of the department for chats, jokes and even sometimes interesting scientific discussions.
On a more professional level, I would like to thank Dr. Tomohiko Matsuo who also taught me a lot about cloning and other technical wonders. Thanks to Chen-Da Kan and Véronique Pauli for their everyday help and support (particularly during this last year); nothing of this would have been possible without you. I would also like to thank Dr. Christoph Bauer, Jérôme Bosset and Mike Parkan from the NCCR Bioimaging platform for their friendliness, their availability and the good advices and help with the confocal microscopes. Finally, I thank Dr. Patrick Descombes and Dr. Olivier Schaad from the NCCR Genomic platform for their help in microarray experiments.
I want and I must be extremely grateful to the staff of the animal facility for their work, taking good care of my mice.
Finally, lots of love to Jennifer and my family for the their support (“Nonna”
thanks for the food and all the rest).
As Luca says: “a few words at the end of a thesis are not enough”. Thank you all again!
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