Table 1. Primer sequences for quantitative real-time PCR. Genes Nlrp2 Nlrp4f Nlrp5 Nlrp9c Nlrp14 b-actin Primer sequences (59-39) Fb: AACACTGAGCCTGAAACACTTGGA Rc: CAGTTCAGTGGAGTGATGGAGCA F: CTTGAACCAGGCAGAGTGCAAC R: TGCCAAATTAAGAACCTTCAACGAC F: CCAGAGCAGGAGCAGACATC R: TTCCAATCCACGTGCTTTCA F: CGCAATTGTACACACATTATCCAAG R: TCAGGCATATAAGTGTTGGTCTCAG F: TTCTCAGCGCAAGGACTTAACTTTC R: AGTCTTTGTCCTCACTCACGGTTTC F: GAAGTGTGACGTTGACATCCG R: ACTTGCGGTGCACGATGGAGG Tanna (6C) 60 60 60 60 60 60 aAnnealing temperature. bForward primer. cReverse primer. doi:10.1371/journal.pone.0030344.t001 rabbit IgG (Beyotime, A0452) for CDX2 and Alexa Fluor 488- labeled goat anti-mouse IgG (Beyotime, A0428) for OCT3/4. The secondary antibodies were diluted 1:500 with PBS containing 1% BSA. After washing in PBS/0.3% PVP, nuclei were stained with DAPI (Beyotime, C1005) for 5 min. Negative controls were determined by processing samples as described above in the absence of primary antibody. Samples were observed using a Nikon eclipse Ti-S microscope (Nikon) or a Zeiss LSM510 confocal microscope equipped with differential interference contrast optics (Carl Zeiss, Inc., Thornwood, NY). Immunogold electron microscopic analysis Ovaries from 10-day-old mice were fixed in 2.5% glutaralde- hyde in 0.1 M phosphate buffer (PB) overnight at 4uC and washed thrice in fresh 0.1 M PB and then transferred into 1% osmium tetroxide in 0.1 M PB for 2 h at 4uC. After dehydrating in graded acetone, the ovaries were embedded in epoxy resin (Durcupan ACM, FLUKA, Buchs, Switzerland) and sectioned (70 nm) with a diamond knife mounted in an Ultracut microtome (Leica, Mannheim, Germany). The sections were mounted on nickel grids for immunogold reactions as described [52]. Briefly, the sections were subjected to blocking buffer and then incubated overnight at 4uC in droplets of rabbit anti-NLRP2 antibody (1:500, Sigma-Aldrich). At the end of incubation, the sections were washed thrice to remove excess primary antibody and incubated again in blocking buffer and then incubated with droplets of the gold-labeled (10 nm) goat anti-rabbit IgG (1:50; Sigma-Aldrich, G7402) for 2 h at RT. After several washings in 0.1 M PB and deionized water, the sections were counterstained with 2% uranyl acetate and lead citrate. Negative controls consisted of samples not incubated with the primary antibody. The sections were observed with a JEM-1230 electron microscope (JEOL Ltd., Tokyo, Japan). GV-stage oocyte and zygote electroporation with custom siRNA Electroporation was used to introduce siRNA into mouse oocytes and zygotes as described [30]. GV-stage oocytes or zygotes were collected and washed in H-KSOM, then incubated in prewarmed acid Tyrode’s solution for 12 s to weaken the zona Nlrp2 is Required for Early Embryonic Development pellucida [29]. They were then washed twice in fresh H-KSOM and kept in the pre-made KSOMaa drops. Afterwards, the siPORT Amine Transfection Agent (Ambion, AM4502, Austin, TX, USA) was diluted in Opti-MEMH I (Invitrogen) medium and incubated for 10 min at RT. The negative control siRNA (Ambion, SilencerH negative control #1, AM4611) or the custom-made Nlrp2 siRNA (mixture of three target-specific 19– 25 nt siRNA designed to knock down mouse Nlrp2 expression; Santa Cruz, sc-149811) was diluted into Opti-MEMH I (Invitro- gen) medium. The diluted negative control siRNA or the diluted custom-made Nlrp2 siRNA were combined with the diluted siPORT Amine Transfection Agent and incubated for 10 min at RT. The GV-stage oocytes or zygotes were mixed with the transfection complexes. Simultaneously, the cell fusion instrument (BTX Inc., ECM 2001; San Diego, CA) was switched on. The GV-stage oocytes or zygotes were linearly arranged in a flat electrode chamber (BTX Inc.) and electroporated with the negative control siRNA (100 nM) and the Nlrp2 siRNA in different concentrations (20, 40 and 60 nM). The electroporation condition was DC 20 volts/1 ms pulse length/3 pulses/0 repeats. The blank siPORTTM electroporation buffer was used as electroporation control (EP control). The negative control siRNA was used as siRNA control (Control siRNA). Following electroporation, the GV-stage oocytes were washed thrice and cultured in MEM media supplemented with 3 mg/ml BSA. The maturation rates of oocytes were calculated after 24 h in vitro culture. Some mature oocytes were processed for qRT–PCR and immunoblotting, others were subjected to parthenogenetic activation and the subsequent development was analyzed. After electroporation, zygotes were washed thrice and cultured in KSOMaa-BSA. Some embryos were collected at 2 h (early 1-cell), 11 h (later 1-cell), 20 h (early 2-cell), 29 h (mid 2-cell) and 38 h (later 2-cell) post-electroporation for qRT–PCR. Some were collected at 4 h (1-cell), 28 h (2-cell) and 52 h (8-cell) post- electroporation for immunoblotting. Construction of NLRP2 expression vector and microinjection Nlrp2 full-length CDS (3141 bp from 75 to 3215) with Sal I and Xma I sites in the 59 and 39 ends was obtained through PCR amplified (sense primer 59–GCAGTCGACCATGGAACATT- TTGATCCCCT–39 antisense primer 59–ATCCCGGGTTA- AAAAATGAAATGAGGAAG–39). The PCR product was di- gested with Sal I and Xma I and inserted to the Sal I/Xma I- digested pIRES2-AcGFP1-Nuc (Clontech) eukaryotic expression vector. The recombinant expression plasmid was then sequenced. Zygotes were placed into a micro-culture dish at RT and microinjected with pIRES2-AcGFP1-Nuc-Nlrp2 (pIRES2-Nlrp2). Microinjection was performed using a Narishige microinjector connected to a micromanipulation system (Nikon, Tokyo, Japan). Approximately 5 pl of 0.8 mg/ml solution of expression plasmid was injected into the cytoplasm of per zygote. Zygotes were kept in H-KSOM during injection and then cultured in KSOMaa-BSA. Zygotes injected with pIRES2-AcGFP1-Nuc (pIRES2) were used as control. Embryos were collected for qRT–PCR and immuno- blotting assays at different stages. TUNEL labeling assay TUNEL (TdT-mediated dUTP nick end labeling) apoptosis assays were carried out with the DeadEndTM Fluorometric TUNEL System (Promega, Madison, WI) according to the manufacturer’s instructions. Apoptosis assays of blastocysts were performed as described [70]. PLoS ONE | www.plosone.org 13 January 2012 | Volume 7 | Issue 1 | e30344