Analysis of gene expression in the bovine blastocyst produced in vitro using suppression-subtractive hybridization

Successful embryonic development is dependant on time and location-specific expression of appropriate genes. Unfortunately, information on stage-specific gene expression during early embryonic development in the bovine is lacking. In the present study, we compared gene expression between in vitro-produced Day 7-8 intact blastocysts (driver) and Day 9-10 hatched blastocysts (tester) using suppression-subtractive hybridization. Pools of 30 embryos for both driver and tester were used in the RNA extraction process. From limited amounts of starting material (similar to400 ng of total RNA), a reverse transcription-polymerase chain reaction (PCR) procedure was used to amplify the mRNA and generate sufficient cDNA to conduct suppression-subtractive hybridization. The subtracted cDNA products were cloned, and 126 cDNAs representing expressed mRNAs were isolated, sized, single-pass sequenced, and compared to known sequences in GenBank. Ninety-two clones provided sequence information for further analysis. Among these, 31 exhibited high homology to known genes. Three, 26S proteasomal ATPase (PSMC3), casein kinase 2 alpha subunit (CK2), and phosphoglycerate kinase (PGK) were selected and further characterized using real-time quantitative PCR to assess their differential expression in hatched blastocysts. Overall, a 1.3-, 1.6-, and 11.5-fold increase in expression level was observed in hatched blastocysts compared with intact blastocyst for PSMC3, CK2, and PGK, respectively. These results show that construction of subtracted cDNA libraries from small numbers of embryos is feasible and can provide information on gene expression patterns during preattachment embryogenesis.