BGEM: An In Situ Hybridization Database of Gene Expression in the Embryonic and Adult Mouse Nervous System

This article describes an open-access gene expression database analyzed for more than 2,000 genes on mouse nervous system tissue in the coronal, sagittal, and transverse orientation representing multiple developmental ages

Selection of hyperfunctional siRNAs with improved potency and specificity

One critical step in RNA interference (RNAi) experiments is to design small interfering RNAs (siRNAs) that can greatly reduce the expression of the target transcripts, but not of other unintended targets. Although various statistical and computational approaches have been attempted, this remains a challenge facing RNAi researchers. Here, we present a new experimentally validated method for siRNA design. By analyzing public siRNA data and focusing on hyperfunctional siRNAs, we identified a set of sequence features as potency selection criteria to build an siRNA design algorithm with support vector machines. Additional bioinformatics filters were also included in the algorithm to increase RNAi specificity by reducing potential sequence cross-hybridization or microRNA-like effects. Independent validation experiments were performed, which indicated that the newly designed siRNAs have significantly improved performance, and worked effectively even at low concentrations. Furthermore, our cell-based studies demonstrated that the siRNA off-target effects were significantly reduced when the siRNAs were delivered into cells at the 3 nM concentration compared to 30 nM. Thus, the capability of our new design program to select highly potent siRNAs also renders increased RNAi specificity because these siRNAs can be used at a much lower concentration. The siRNA design web server is available at http://www5.appliedbiosystems.com/tools/siDesign/

Early Embryonic Lethality in Mice with Targeted Deletion of the CTP:Phosphocholine Cytidylyltransferase α Gene (Pcyt1a)

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-controlling step in the biosynthesis of phosphatidylcholine (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

The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo

Exosomes are small (30–150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication—exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform

Walk-through experiment results for 78 ATS siRNA duplexes.

<p>Results of walk-through experiments measured at day 6 post transfection with synthetic siRNA duplexes using EGFPB reporter cell line. (<b>A</b>) Clustered heatmap to show % gain in EGFP signal conferred by 78 ATS siRNA duplexes, tested as singles. (<b>B</b>) Clustered heatmap to show % gain in EGFP signal conferred by siRNA duplexes segregated into three pools that of duplexes active as singles, inactive as singles or with all inclusive. Rep stands for replicate, AVG stands for average of the four replicates.</p

Schematics for Alternate Targeting Sequence Generator (ATSG).

<p>Hairpin inside the cell gets cleaved at its theoretical site and silences its target specifically. Inefficiencies in cleavage would lead to ATSG, generating random targeting sequencing which silence alternate targets, making it extremely difficult to comprehend the eventual phenotypic outcomes.</p

List of 27 total cell lines used towards confirmation of 7 gene-signature knockdowns.

1<p>Reporter cell line used in the screen and walk-through experiments.</p>2<p>Cell line used for TRCN#40273 validation performed by Sigma-Aldrich in collaboration with the Broad Institute.</p><p>wt; wild-type, mut; mutant.</p

mRNA knockdown profiles for 7 gene-signature post transduction with TRCN#40273.

<p>(<b>A</b>) qRT-PCR results for panel of 4 cell lines including reporter cell line. (<b>B</b>) qRT-PCR results for panel of 6 melanoma cell lines. (<b>C</b>) qRT-PCR results for panel of 6 adenocarcinoma cell lines. (<b>D</b>) qRT-PCR results for panel of 5 cell lines derived from breast, kidney or retina. (<b>E</b>) Clustered heatmap to show mRNA knockdown levels of 7 genes across 27 distinct cell lines. (<b>F</b>) qRT-PCR results for 3 <i>DICER1</i>^mut and 3 <i>DICER1</i>^wt cell lines. Data in the bar graphs is expressed as average ± standard error.</p