incubating the membranes for 30 min in 0.1 M glycine buffer (pH 2.5) followed by a 30 min incubation in 0.1 M Tris buffer containing 2% SDS and subsequently washing three times in TBST. Immunocytochemistry and Histology Spread-preparations of meiocytes were produced as described by de Boer et al. [59] and in each experiment spread-preparations were immunostained at the same time with the same mixture of the appropiate affinity-purified primary antibodies: rabbit anti- SYCE3 (1:50), guinea pig anti-SYCE3 (1:100), guinea pig anti- SYCE1 (1:1000) [29], guinea pig anti-SYCE2 (1:200) [29], guinea pig anti-SYCP1 (1:150) [60], rabbit anti-SYCP1 (1:200) [26], guinea pig anti-SYCP3 (1:150) [61], rabbit anti-SYCP3 (1:200; NB300-232, Acris, Herford, Germany) mouse anti-cH2AX (1:500; 05-636, Millipore, Schwalbach/Ts., Germany), mouse anti-RPA (1:40; NA19L, Calbiochem, Darmstadt, Germany), rabbit anti- RAD51 (1:30; PC130, Calbiochem), mouse anti-MLH1 (1:30; 551091, BD Pharmingen, Heidelberg, Germany). Secondary antibodies were applied as specified by the manufacturer (Dianova). Histology was performed on 5 mm sections of paraffin-embedded testis or ovary tissue fixed overnight in 4% formaldehyde according to standard protocols. Staging of mouse seminiferous tubule cross- sections was done according to Ahmed and de Rooij [62]. A TUNEL assay was carried out on 10 mm sections of paraffin embedded testis or ovary tissue fixed overnight in 4% formaldehyde using the ApopTag Peroxidase In Situ Apoptosis Kit (Millipore) according to the manufacturer’s protocol. Statistical Analysis Statistically significant differences in the medians of RPA and MLH1 foci comparing Syce32/2 and control cells were verified by Mann-Whitney U test. Numbers of pachytene and diplotene stages from control and Syce32/2 19.5 dpc oocytes were statistically compared using a chi-square test. Microscopy and Imaging Fluorescence microscopy was carried out by using a Zeiss Axiophot fluorescence microscope (Zeiss, Munich, Germany) equipped with a Plan-NEOFLUAR 406/0.75 or a Plan-NEO- FLUAR 206/0.5 objective and the AxioCam MRm (Zeiss) camera. Digital images were pseudocoloured using the AxioVs40 V4.7.1.0 software release and processed using Adobe Photoshop (Adobe Systems, San Jose, CA). Light microscopy was carried out using the stereo microscope MZ FLIII (Leica). Confocal laser scanning microscopy was performed with a Leica TCS-SP2 confocal laser scanning microscope (Leica, Bensheim, Germany) equipped with a 636/1.40 HCX PL APO lbd.BL oil-immersion objective. All confocal images are pseudocoloured using the Leica TCS-SP2 software and are two-dimensional projections calculated from a series of sequenced optical sections using the maximum projection algorithm (Leica). Imaging of wild-type and mutant SYCE1, SYCE2, SYCP1, SYCP3 and SYCE3 cells was performed using the same microscope settings. Digital images were processed with the same settings in Adobe Photoshop (Adobe Systems). Electron Microscopy Electron microscopy was performed using ultra thin sections of testis tissue fixed in 2.5% glutaraldehyde and 1% osmium tetroxide as described previously [22]. For immunoelectron microscopy 10 mm cryosections of rat testis were fixed with acetone for 10 min at 220uC and air-dried. Incubation with Novel Synaptonemal Complex Protein SYCE3 primary antibodies (guinea pig anti-SYCE3 (1:500–1:1500); mouse anti-SYCP1 (1:50) [26] was carried out in a humidified box for 4 h at room temperature. After rinsing twice in PBS, sections were fixed for 10 min in 2% formaldehyde and blocked with 50 mM NH4Cl. 6 nm gold conjugated secondary antibodies were incubated overnight at 4uC and samples were washed subsequent- ly in PBS. Samples were fixed for 30 min in 2.5% glutaraldehyde and postfixed in 2% osmium tetroxide. After rinsing three times with H2O, samples were dehydrated in an ethanol series and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate according to standard procedures [22]. Generation of Syce32/2 Mice We deleted the Syce3 gene by replacing the entire coding sequence of SYCE3 (exons 2 and 3) with a neomycin cassette in reverse orientation using a modified pKSloxPNT vector. The vector for homologous recombination was designed as follows (see also Figure S2A): a 1.1 kbp genomic fragment (F1) containing part of intron 1 was cloned into the SalI restriction site downstream of the neomycin cassette and a 4.1 kbp fragment (F2) containing part of intron 3 was ligated into the EcoRI restriction site located in between the thymidine kinase and neomycin cassette. Electropo- ration of the modified replacement vector into the R1/E embryonic stem cells, laser assisted microinjection into 8-cell C57BL/6 morula and transfer of morula into CD1 (outbred) foster mice was performed at the transgenic core facility of the Max Planck Institute of Molecular Cell Biology and Genetics, Dresden. After electroporation and selection, we identified one positive ES cell clone by PCR with external primers (oligonucleotide sequence for genotyping: see Table S2) and confirmed correct targeting by Southern blot. For Southern blot analysis 10 mg BstEII digested DNA derived from ES cells (or tail tips of Syce3+/+, Syce3+/2 and Syce32/2 mice) was loaded on a 0.8% agarose gel, subsequently transferred to a nylon membrane and correct insertion was tested with both external and neomycin probes. Blastocyst injection of the ES cell clone produced germline transmitting chimeras. Mating of chimeras with C57BL/6 mice gave rise to wild-type and Syce3+/2 mice. Intercrossing of Syce3+/2 mice produced offspring with all genotypes in Mendelian ratio. To confirm the absence of SYCE3 we performed PCR, Southern blot, immuno- fluorescence analysis on spread Syce32/2 spermatocytes and Western Blot analysis on Syce32/, testis tissue with polyclonal antibodies raised against the full-length SYCE3 protein (data not shown and Figure S1A). Transfection and Co-Immunoprecipitation In order to express full-length SYCE3, SYCE1, SYCE2, Tex12 as well as SYCP1 N-terminal (aa 1–120) and SYCP1 C-terminal (aa 820–997) fusion constructs in the culture cell line COS-7 (green monkey kidney) for co-immunoprecipitation analysis, the respective cDNAs were inserted into pEGFP (Clontech, Heidel- berg, Germany) or pCMV-Myc (BD Bioscience) vectors [60]. The fusion-constructs used (including the oligonucleotides used for cloning) are summarized in Table S3. Cells were transfected with the respective constructs using the effectene system according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Co- immunoprecipitation experiments were performed as described by Stewart-Hutchinson et al. [48] with the following modifications: (1) myc and EGFP constructs were immunoprecipitated with 0.5 mg of mouse anti-myc (R950-25, Invitrogen) or 0.5 mg of mouse anti- GFP (sc-9996, Santa Cruz Biotechnology) antibody per 60-mm dish. (2) Immune complexes were pulled down by protein G dynabeads (100-03D, Invitrogen). PLoS Genetics | www.plosgenetics.org 13 May 2011 | Volume 7 | Issue 5 | e1002088