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The genetic alternations in human medulloblastoma (MB) include mutations in the Sonic hedgehog (SHH) signaling pathway and TP53 inactivation. Studies revealed that in tumor cells purified from double heterozygotes, Ptc1 allele, but not Ink4c was inactivated and hence it was concluded that Ink4c combined with Ptc1 mutation is haploinsufficient for tumor suppression.

The regulation of the Clara cell 10 kilodalton protein (CC10) gene expression by interferon-gamma was analyzed in Clara cell lines that express CC10 and hepatocute nuclear factor 3beta (HNF-3beta). In vivo and in vitro administration of recombinant murine IFN-gamma in HFN-3beta- and CC10-expressing Clara cell lines increased the expression of CC10 messenger RNA. The effects of INF-gamma on CC10 gene expression also indicated the presence of a regulatory pathway between the proinflammatory cytokine and Clara cells during pulmonary inflammation.

CTP:pbosphocholine cytidylyltransferase (CCT) catalyzes a rate-controlling step in the biosynthesis of phosphatidyleholine (PtdCho). Multiple CCT isoforms, CCTα, CCTβ2, and CCTβ3, are encoded by two genes, Pcyt1a and Pcyt1b. The importance of CCTα in mice was investigated by deleting exons 5 and 6 in the Pcyt1a gene using the Cre-lox system. Pcyt1a-/- zygotes failed to form blastocysts, did not develop past embryonic day 3.5 (E3.5), and failed to implant. In situ hybridization in E11.5 embryos showed that Pcyt1a is expressed ubiquitously, with the highest level in fetal liver, and CCTα transcripts are significantly more abundant than transcripts encoding CCTβ or phosphatidylethanolamine (PtdEtn) N-methyl transferase, two other enzymes capable of producing PtdCho. Reduction of the CCTα transcripts in heterozygous E11.5 embryos was accompanied by upregulation of CCTβ and PtdEtn N-methyltransferase transcripts. In contrast, enzymatic and real-time PCR data revealed that CCTβ (Pcyt1b) expression is not upregulated to compensate for the reduction in CCTα expression in adult liver and other tissues from Pcyt1a+/- heterozygous mice. PtdCho biosynthesis measured by choline incorporation into isolated hepatocytes was not compromised in the Pcyt1a+/- mice. Liver PtdCho mass was the same in Pcyt1a+/+ and Pcyt1a+/- adult animals, but lung PtdCho mass decreased in the heterozygous mice. These data show that CCTα expression is required for early embryonic development, but that a 50% reduction in enzyme activity has little detectable impact on the operation of the CDP-choline metabolic pathway in adult tissues. [ABSTRACT FROM AUTHOR]

Presents information on the Clara cell (CC) line, to analyze the regulation of the CC10 gene by interferon- (IFN-). Primary function of the lung; How the lung functions efficiently in gas exchange; Pivotal molecules in the regulation of lung immune responses; Details on the study.

Discusses a study that used bacterial artificial chromosomes to dose Zipro1, which encodes a zinc-finger transcription factor in mice. Discovery that Zipro1 regulates cell growth; Enhancement of cell proloferation in the cerebellum and skin.

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment is associated with major long-term side effects; therefore, new nontoxic therapies, targeting specific molecular defects in this cancer, need to be developed. We use a mouse model of medulloblastoma to show that inhibition of the Sonic Hedgehog (Shh) pathway provides a novel therapy for medulloblastoma. A small molecule inhibitor of the Shh pathway, HhAntag, blocked the function of Smoothened in mice with medulloblastoma. This resulted in suppression of several genes highly expressed in medulloblastoma, inhibition of cell proliferation, increase in cell death and, at the highest dose, complete eradication of tumors. Long-term treatment with HhAntag prolonged medulloblastoma-free survival. These findings support the development of Shh antagonists for the treatment of medulloblastoma. [Copyright &y& Elsevier]

Aim: To develop protocols for isolation of exosomes and characterization of their RNA content., Methods: Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight(®) analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology., Results: Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA., Conclusion: We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques.

RNA interference (RNAi) is a mechanism by which the introduction of small interfering RNAs (siRNAs) into cultured cells causes degradation of the complementary mRNA. Applications of RNAi include gene function analysis, pathway analysis, and target validation. While RNAi experiments have become common practice in research labs, multiple factors can influence the extent of siRNA-induced knockdown (and thus biological outcome). A properly designed and selected siRNA sequence, siRNA modification format, choice of transfection reagent/technique, optimized protocols of siRNA in vitro delivery, and an appropriate and optimized readout are all critical for ensuring a successful experiment. In this chapter, we describe a typical in vitro siRNA experiment with optimization of transfection conditions and analysis of siRNA potency, i.e., mRNA knockdown with quantitative real-time PCR.

RNA interference (RNAi) is a regulatory mechanism of eukaryotic cells that uses small interfering RNAs (siRNA) to direct homology-dependent control of gene activity. Applications of RNAi include functional genomics, in vivo target validation, and gene-specific medicines. A key to in vivo application of siRNA is the advancement of efficient delivery to organs, tissues, or cell types of interest. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (post-transfection) and in animals (post- injection). We have adopted the Applied Biosystems TaqMan(®) based stem-loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. In this chapter, application protocols are described, which enable robust quantification of siRNA, including chemically modified molecules, in vitro and in vivo.

Despite the promise of short interfering RNAs (siRNA), contending with off-target is a challenge for RNAi users. To alleviate these problems, we have developed locked nucleic acid (LNA) modified siRNAs and optimized performance using cellular phenotypic assays as well as microarray analysis. During development, we compared LNA and 2'OMethoxy (2'OMe) chemistries placed strategically throughout the siRNA molecule and found a novel pattern of LNA placement that greatly improved the specificity of the siRNA and reduced it's toxicity in culture while preserving the potency of the siRNA. The improvements in specificity made by LNA-modified siRNAs were developed and validated by measuring the phenotypic signatures in a high content cell-based screening assay as well as comparison of the level of differentially expressed genes observed in microarray analysis between modified and unmodified siRNAs. HT screening of a collection of genes demonstrated that the LNA-modified siRNAs exhibits the best overall rate to elicit the expected phenotype, reduced toxicity and achieved an improved coherence of phenotype compared to 2'OMe-modified or unmodified siRNAs.