PARENT SESSION
Oocyte Development
90
MICROARRAY STUDY TO IDENTIFY GENE EXPRESSION ASSOCIATED EXCLUSIVELY TO OOCYTE.
Robert, Claude1, Gagné, Dominic1, McGraw, Serge 1, Bousquet, Daniel2, Barnes, Frank3, Sirard, Marc-André1, 1 2 3
ABSTRACT- This study was conducted to identify mRNA exclusively found in the oocyte. The main objective is to better understand the molecular mechanisms underneath unique features of the oocyte such as the capacity of the oocyte cytoplasm to reprogram a differentiated nucleus. The microarray technology was used to perform high throughput screening of a large amount of isolated candidate clones. The clones were collected from the end products of 4 cDNA library subtractions. Two subtractions were done to compare different classes of bovine oocytes. The third subtraction was performed using 700 bovine oocytes subtracted with a pool of several tissues (liver, kidney, muscle, theca and granulosa cells) while the last subtraction was performed on different types of granulosa cells. All the subtractions were performed by PCR using the PCR Select kit (Clontech, Palo Alto, CA). An average of 900 clones was collected from each subtraction. A total of 3650 clones were printed on CMT-GAPS coated glass slides (Corning, Corning, NY) using a ChipWriter robot (ESI, Toronto, ON). Probes were prepared from the subtracted cDNA products resulting from the third subtraction. Generally, when using unsubtracted probes, low expressing genes are often missed because they produce insufficient signal. However, the subtracted end products are enriched in differentially expressed cDNAs and this normalization should be useful to identify genes that are expressed at low levels. The probes were prepared by random labeling using the Klenow enzyme (Roche, Laval, QC) to incorporate Cy-3 or Cy-5 (NEN, Boston, MA). The signals were strong over the entire microarray and the background was low. By considering a 5-fold intensity ratio to be significantly different, 14 clones were isolated as specific to the oocyte. Sequencing is needed to identify the clones and tissue specificity must be confirmed by quantitative RT-PCR. This project was supported by the Natural Sciences and Engineering Research Council of Canada and Semex Canada.
KEY WORDS: microarray, oocyte, gene expression, bovine