The finding that significant fetal lung injury occurred silently before preterm labor is also novel and quite sobering for the development of preventive strategies

c tag was inserted into the unique ClaI site of pGEM-PrP; ClaI. Two synthetic 59-phosphorylated oligonucleotides were annealed to produce a double-stranded DNA with 59-protruding, ClaI compatible ends. The myc-fwd oligonucleotide sequence encodes the human myc epitope, EQKLISEEDL. The myc-tag was ligated into ClaI digested pGEM-PrP; ClaI generating pGEMPrP-myc; ClaI. Finally, the Oritavancin (diphosphate) custom synthesis XmaI-PmlI fragment of phgPrP was replaced by the XmaI-PmlI fragment of pGEMPrP-myc; ClaI yielding plasmids phgPrP-myc and the construct was verified by sequencing. B6;129S5-Prnpo/o mice. To differentiate PrPmyc transgenic littermates with Prnp+/o and Prnpo/o genotype the presence of the endogenous Prnp+ allele was tested by PCR analysis using primers Prnp intron 2 and P10rev amplifying 11741928 a 352 bp product for the Prnp wild-type allele but no PCR product for the Prnpo allele. For Northern blot analyses, RNA was extracted using Trizol. A randomly 32P-labeled restriction fragment encompassing all of exons 1 and 2, all of the ORF and a part of exon 3 was used as a PrP probe. This probe hybridizes with all wild-type and tagged PrP mRNAs as well as the readthroughRNA from the disrupted Prnp locus. Southern blot analyses were performed using a 640 bp DNA probe synthesized by incorporation of digoxigenin-11-dUTP during PCR using PrP-specific primers and hybridization was performed following established protocols. For the actin control the Northern blot was probed with an inhouse generated mouse beta-actin probe cloned from full-length cDNA. Rescue of Shmerling’s disease o=o PrPmyc mice were crossed with 25279926 PrPDF mice to obtain double transgenic animals with Prnpo/o genotype needed for the experiment described in Fig. 1. Animals were examined twice each week for symptoms of cerebellar dysfunction, including ataxia, tremor, weight loss, rough hair coat, and kyphosis. Scoring of neurological signs was performed according to a four-degree clinical score system and mice were euthanized within 3 days of reaching a score of 3.5. Generation and characterization of transgenic mice The phgPrP-myc plasmid, driven by the endogenous Prnp promoter in the context of the PrP half-genomicconstruct , was digested with NotI and SalI to remove its prokaryotic backbone. Pronuclear injections were performed into fertilized oocytes derived from a B6D2F16B6;129S5-Prnpo/o mating.To obtain PrPmyc transgenic animals on a Prnpo/o knockout background, the founders were backcrossed to homozygous Western blot analyses Homogenates of noninfectious brain and spleen were prepared in sterile PBS/0.5% Nonidet P-40 and protease inhibitors by repeated extrusion through syringe needles of successively smaller size. Homogenates of infectious brains were generated using a rhybolyzer in a Interactome of Myc-Tagged PrP biosafety level 3 laboratory. After centrifugation for 10 min at 2’400 rpm at 4uC, supernatant was loaded onto 12% SDSpolyacrylamide gels. Proteins were transferred to nitrocellulose membranes by wet blotting, and first exposed to mouse monoclonal anti-PrP antibody POM-1, 1:10’000 or mouse monoclonal anti-myc antibody 4A6, then to peroxidase-labeled rabbit antimouse antiserum and developed using the ECL detection system. Antibody incubations were performed in 1% Top Block in PBS-Tween for 1 hour at room temperature or overnight at 4uC. The same protocol was applied to generate Western blots shown in Fig. 4 DF using anti-M6-7 antibody diluted 1:5000, anti-CNPase antibody diluted 1:500 and antiNeurofasci

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