International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

D12. Large Coverage of the Mouse Genome with Cap-Selected Full-Length cDNAs

Piero Carninci1, Kazuhiro Shibata1, 2, Masayoshi Itoh1, 2, Hideaki Konno1, 2, Yuko Shibata1, Kenjiro Sato1, Norihito Hayatsu1, Toshiyuki Shiraki1, Tomoko Hirozane1, Katsunori Aizawa1, 2, Hidemasa Bono1, Koji Kadota1, Shinji Kondo1, Jun Kawai1, 2, Atsushi Yoshiki3, Moriaki Kusakabe3, Masami Muramatsu1, 2, Yasushi Okazaki1 and Yoshihide Hayashizaki1, 2
1Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences
Center (GSC) and Genome Science Laboratory, RIKEN Tsukuba Institute,
Experimental Animal Facility RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba 305-0074, Japan

Target of our project is to isolate at least one full-length cDNA of every mouse gene in order to create a reference cDNAs set for mammalians. Full-length cDNA allows all subsequent functional characterization such as prediction of protein structure and function, protein-protein interaction, structural investigation, expression analysis, protein expression, etc.

Gene discovery based on one-pass sequencing of Cap-trapper full-length cDNA libraries has been over years very demanding. In fact, cloning efficiency of short cDNA is largely more effective than long cDNAs. Cloning rarely expressed genes and genes specifically expressed in restricted tissues constituted a technical challenge.

To address these problems, we have developed techniques for the construction of large insert size, normalized/subtracted full-length cDNA libraries even from microdissected tissues that do not involve the use of PCR. We have recently developed techniques such as multiple subtractions to remove from libraries the cDNAs that have already been isolated.

The identification of tissues in which genes are differentially expressed is another important aspect of the project. Preparation of cDNA libraries form tissues of pathological interest (such as cancer, inflammation and other disease models) allows to isolate at high efficiency new cDNAs that are also very important, being potentially responsible for diseases.

To facilitate functional analysis, we have also developed cloning vectors capable to transfer full-length cDNA inserts without subcloning into other functional vectors. We also modified the cloning of full-length cDNA and their complete 5' UTRs without using homopolymeric tails.

To the date of the submission of this abstract, we estimate that our clones, deriving from full-length cDNA libraries, cover about 60% of mouse genes. At the moment, more than 90% of Unigene clones contain a sequence from our clones, and a large part is constituted only from our clones. Our analysis based on the currently available data suggests also that the number of mouse genes exceeds 100,000.


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