Akira Watahiki

Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage

We introduce cap analysis gene expression (CAGE), which is based on preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5′ end mRNAs. CAGE allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage analysis. By analyzing four libraries (brain, cortex, hippocampus, and cerebellum), we redefined more accurately the TSPs of 11-27% of the analyzed transcriptional units that were hit. The frequency of CAGE tags correlates well with results from other analyses, such as serial analysis of gene expression, and furthermore maps the TSPs more accurately, including in tissue-specific cases. The high-throughput nature of this technology paves the way for understanding gene networks via correlation of promoter usage and gene transcriptional factor expression.

Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas

It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line–specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

The DT40 web site: sampling and connecting the genes of a B cell line

Thousands of new vertebrate genes have been discovered and genetic systems are needed to address their functions at the cellular level. The chicken B cell line DT40 allows efficient gene disruptions due to its high homologous recombination activity. However, cloning the gene of interest is often cumbersome, since relatively few chicken cDNA sequences are present in the public databases. In addition, the accumulation of multiple mutations within the same cell clone is limited by the consumption of one drug-resistance marker for each transfection. Here, we present the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40.html), which includes a comprehensive database of chicken bursal ESTs to identify disruption candidate genes and recyclable marker cassettes based on the loxP system. These freely available resources greatly facilitate the analysis of genes and genetic networks.

Trehalose-enhanced isolation of neuronal sub-types from adult mouse brain

Efficient isolation of specific, intact, living neurons from the adult brain is problematic due to the complex nature of the extracellular matrix consolidating the neuronal network. Here, we present significant improvements to the protocol for isolation of pure populations of neurons from mature postnatal mouse brain using fluorescence activated cell sorting (FACS). The 10-fold increase in cell yield enables cell-specific transcriptome analysis by protocols such as nanoCAGE and RNA seq.

RNA extraction from sorted neuronal subtypes

Protocol Summary Efficient isolation of specific, intact, living neurons from the adult brain is problematic due to the complex nature of the extracellular matrix consolidating the neuronal network...