PARENT SESSION
DEVELOPMENT OF THE EMBRYO
Harborside C
7:30 AM-10:00 AM

(150) CHARACTERIZATION OF cDNA LIBRARIES DEVELOPED FROM PORCINE OOCYTES AND EMBRYOS: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES.

Whitworth, Kristin¹, Lucy, Mathew¹, Ries, James², Springer, Gordon², Guillen, Arturo², Forrester, Lawrence³, Mathialagan, Nagappan⁴, Didion, Brad⁴, Prather, Randall¹, Green, Jonathan¹, ¹ Dept of Animal Sciences, Columbia, MO² Dept of Computer Engineering and Computer Science, Columbia, MO³ Molcular Biology Program, Columbia, MO⁴ Agricultural Sector, Dairy Business, St. Louis, MO

ABSTRACT- The refinement of in vitro techniques for the production of embryos has improved the efficiency in which viable embryos can be generated for experimental purposes (e.g., cloning). Despite decades of research on oocytes and embryos, there is currently little known about the genetic events that take place during early development. To better understand such events, we have recently initiated a large-scale cDNA sequencing project to provide molecular information regarding the transcripts expressed by porcine oocytes, 4-cell stage embryos (in vivo and in vitro produced) and blastocytes (in vivo and in vitro produced). The plasmid cDNA libraries were generated by using a PCR based amplification of reverse-transcribed mRNA from each source. Each library was characterized as to the number of clones, average insert size and extent of genomic, mitochondrial and ribosomal RNA contamination. Over two thousand sequencing attempts were performed on each library (10,848 total), resulting in 8,661 ESTs. Analysis of the sequences by clustering algorithms revealed 2,576 unique sequences, of which 1,188 have yet to be identified from other sequencing projects, i.e., they had a Blast score of less than 200 when the GenBank and TIGR databases were searched. Sequencing of the libraries identified numerous changes in expression of several of the sequence tags at different stages of development and between the in vivo and in vitro produced embryos. Many of the differentially expressed clones were uncharacterized cDNA that either were not present in the sequence databanks or only matched tags from other EST sequencing projects. The identification and characterization of such differentially expressed genes provides a preliminary view of those genes whose transcription is markedly up- or down-regulated during early development. Such results also define those genes that are aberrantly expressed due to in vitro culture. Supported by a grant from Monsanto Co.

KEY WORDS: Embryo, In vitro culture, cDNA