24 GLOBAL TRANSCRIPT PROFILING OF CLONED BOVINE BLASTOCYSTS USING AFFYMETRIX GENECHIP TECHNOLOGY

Abstract

Identification of genes implicated in the biological processes of somatic cell nuclear transfer will improve our understanding of reprogramming events, i.e. the transformation of a lineage-committed cell into a pluripotent one. In addition, the gene expression profile of cloned embryos can help explain the widely reported developmental failures in cloned animals. In this study, we investigated global gene expression profiles of bovine in vitro-fertilized and cloned embryos using Gene Chip Bovine Genome Arrays (Affymetrix, Inc., Santa Clara, CA, USA). For the generation of cloned bovine blastocysts from two adult fibroblast lines (C and D), we employed methods previously proven to generate live offspring and compared these offspring to in vitro-produced blastocysts. Total RNA isolated from groups of 10 blastocysts was amplified by a template-switching PCR. Amplified cDNAs were used to synthesize biotin-labeled antisense RNAs (aRNAs) during and in vitro transcription reaction. Labeled aRNAs were hybridized to microarrays as described by the manufacturer. Experiments were performed in four replicates. Expression data were analyzed using the Significance Analysis of Microarrays (SAM; Tusher et al. 2001 Proc. Natl. Acad. Sci. 98, 5116-5121) procedure and software. Overall, 48.4% and 46% of 23 000 bovine transcripts spotted on the arrays were present in cloned and in in vitro-produced control blastocysts, respectively. The SAM procedure identified 43 genes that changed at least 1.5-fold, with an estimated false discovery rate (FDR) of 20%. Comparison of gene expression between NT embryos produced from two different cell lines and IVF controls with the same criteria revealed 6 (clones from cell line C vs. IVF) and 46 (clones from cell line D vs. IVF) differentially expressed genes. The number of transcripts expressed differentially between the cloned embryos with different donor cell origin was 437. Of the 43 differentially expressed transcripts in cloned blastocysts, 13 have unknown functions and the rest of the genes related to cell structure (tuftelin, desmoplakin), cell cycle/mitosis (Kinesin like 4, katanin, stathmin, PCNA), energy metabolism (lactate dehydrogenase, ATPsynthase, lipid-binding protein, keto acid dehydrogenase E1, metallothionein), and cell signaling (GTP-binding protein1, GTP binding stimulatory protein). Our results indicate that expression profiles of cloned blastocysts could be affected by somatic donor cell.