International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

I6 Gene Expression Profiling Using Mouse Full-Length 20K cDNA Microarray

Yasushi Okazaki1, Rika Miki1,2, Yosuke Mizuno1,2, Yasushiro Tomaru1,3 Kouji Kadota1, Piero Carninci1, Kazuhiro Shibata1,4, Masayoshi Itoh1,4, Yasuhiro Ozawa1, Jun Kawai1,4, Hideaki Konno1,4, Yoshifumi Fukunishi1,4, Toshinori Kusumi1, Hitoshi Goto1,5, Hiroyuki Nitanda1,5, Youhei Hamaguchi1,6, Itaru Nishizuka1.6, Masami Muramatsu1,4, Jun Yoshiki7, Moriaki Kusakabe7, Josephe DeRisi8, Vishy Iyer8, Michael Eisen8, Patrick O. Brown8, and Yoshihide Hayashizaki1,2,4. 1Laboratory for Genome Exploration Research Project, Genomic Sciences Center (GSC) and Genome Science Laboratory, Tsukuba Life Science Center, the Institute of Physical and Chemical Research (RIKEN); 2Tsukuba University; 3Espec Oligo Service Corporation; 4CREST, Japan Science and Technology Corporation (JST); 5Tohoku University; 6Yokohama University; 7Development of Experimental Animal Research, Tsukuba Life Science Center, RIKEN; 8Stanford University

The goal of the Genome Science Laboratory of RIKEN is to clone and sequence the largest possible number of full-length mouse cDNAs and then to sequence these full-length cDNAs. We have constructed over 80 libraries from embryonic tissues of different developmental stages and adult tissues in order to ensure the greatest possible coverage of the expressed mRNA. The strategy for the construction of full-length cDNAs will be presented in the other section.

More than 250,000 successful sequencing passes have been performed with the use of two in house developed tools; a high-throughput plasmid preparation system and the RISA 384 capillary sequencer. Most of the sequences were performed from 3' end in order to select individual cDNAs. We have selected more than 30,000 different cDNAs.

Using these sets of RIKEN full-length cDNA, we have established Gene Expression Microarrays containing 20 K set of RIKEN full-length cDNA unique mouse genes (http://genome.rtc.riken.go.jp) These set have been used to profile expression patterns of various adult and embryonic tissues. Target DNAs were PCR amplified and printed on the Poly-L-lysine coated slide glasses. Target DNAs were blocked by excess amount of Cot1DNA. Probes were labeled by two-color fluorescent dye using random primer and reverse transcriptase. Normalization has been achieved using a global normalization method. We have also developed a program to filter the noise. The experiment was done twice and the reproducible results were extracted and clustered. We will present a large set of database, which show the spatial, and temporal expression patterns of mice. These mouse full-length 20 K cDNA microarrays are widely applicable to analyze the global expression profiling of normal and diseased status of the mice.

 


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