| Developmental Genomics and Aging Section |
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| Minoru S.H. Ko, M.D., Ph.D., Chief Senior Investigator |
| 1. Mouse Embryonic cDNA Clones and Microarrays |
| A catalog of genes in the form of cDNA clones is the complement to the sequence of genomes, providing not only the confirmation of predicted gene structures, but also the materials for cDNA microarrays and for functional analyses or proteomics. Primary means of achieving this goal have been expressed sequence tag (EST) projects, which essentially comprise single-pass sequencing of randomly picked cDNA clones. One major difficulty to construct a cDNA library from early embryonic materials is the scarcity of the starting materials. We have recently developed a novel design of linker-primer that allows one to amplify differentially long tracts (average 3.0 kb with size ranges of 1 - 7 kb) or short DNAs (average 1.5 kb with size ranges of 0.5 - 3 kb) from a complex mixture. The method allows one to generate cDNA libraries enriched for long transcripts without size selection of insert DNAs. All our recent cDNA libraries have been made by this new method, and thus, a significant fraction of these cDNA clones contains complete open reading frame ("full-length"). We have thus far generated ~140000 ESTs from early mouse embryos and mouse stem cells (http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html). |
| To use these mouse cDNA clones for the cDNA microarray applications, we have selected ~15,000 unique mouse cDNA clones from ~52,000 cDNA clones and rearrayed them into 96-well microtiter plates. The clone set, called "NIA 15K Mouse cDNA Clone Set," consists of 2132 genes from E7.5 extraembryonic tissue cDNA library, 893 genes from E7.5 embryonic tissue cDNA library, 7692 genes from preimplantation cDNA libraries (unfertilized eggs, fertilized eggs, 2-cell, 4-cell, 8-cell, 16-cell embryos and blastocysts), and 4620 genes from E12.5 mesonephros and newborn ovary cDNA libraries. In May 2000, we distributed the clone set free-of-charge to 8 academic centers, all of which in return agreed to redistribute the clone set to at least 10 other research institutions at a nominal charge. The system has worked out well and more than 100 microarray facilities worldwide are using the NIA 15K Mouse cDNA Microarray (http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html). We are currently working on the production of additional 11,000 cDNA clones ("NIA 11K Mouse cDNA Clone Set"). |
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