Submitted to: Animal Genetics International Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: June 1, 2000
Publication Date: July 22, 2000
Citation: Sonstegard, T.S., Capuco, A.V., Van Tassell, C.P., Wells, K.D., Ashwell, M.S. 2000. Characterization of a normalized cdna library constructed from bovine mammary gland tissues [abstract]. Animal Genetics International Conference Proceedings. Technical Abstract: Characterization of mammary-specific gene expression will aid the discovery of biological factors that influence production and udder health in dairy cattle. Expressed sequence tags (ESTs) are a critical component of this investigation, serving as a resource for gene discovery, mapping, and expression profiling. As a resource to generate 10,000 unique bovine mammary ESTs, we constructed a Cot500 normalized cDNA library. To encompas a larger repertoire of gene expression, mRNA was isolated from udder biopsies performed on animals from eight different stages of mammary growth, development, and health. Equimolar amounts of mRNA from each stage were combined and used to synthesize cDNA. To make the library amenable for automated high-throughput sequence analysis, 50,000 clones were picked and arrayed into 384 well plates. DNA template for sequencing was generated by PCR amplification of cDNA inserts from bacterial culture. A preliminary analysis of the sequencing data from 4,224 clones was performed using BLAS against GenBank nr and dbEST to assess inter- and intra-library EST redundancy. Clonal redundancy within the library was 35%. Of the uniquely identified ESTs, 61% had no similarity to bovine sequence, and 16% had no identity to any sequence. For the ESTs (39%) with similarity to bovine sequence, 62% of these were redundant with ESTs generated from the four bovine cDNA libraries at ARS-USDA MARC. The EST sequence data indicate ~16,000 clones need to be sequenced to reach our goal of 10,000 ESTs.