title: Gene expression profiling of individual bovine nuclear transfer blastocysts.
abstract: During somatic cell nuclear transfer the gene expression profile of the donor cell has to be changed or reprogrammed extensively to reflect that of a normal embryo. In this study we focused on the switching on of embryonic genes by screening with a microarray consisting of 5000 independent cDNA isolates derived from a bovine blastocyst library which we constructed for this purpose. Expression profiling was performed using linearly amplified RNA from individual day 7 nuclear transfer (NT) and genetically half-identical in vitro produced (IVP) blastocysts. We identified 92 genes expressed at lower levels in NT embryos whereas transcripts of 43 genes were more abundant in NT embryos (P < or = 0.05, > or = 1.5-fold change). A range of functional categories was represented among the identified genes, with a preponderance of constitutively expressed genes required for the maintenance of basal cellular function. Using a stringent quantitative SYBR-green real time RT-PCR based approach we found, when comparing the means of the expression levels of a larger set of individual embryos, that differences were small (< 2-fold) and only significant for two of the seven analysed genes (KRT18, SLC16A1). Notably, examination of transcript levels of a single gene in individual embryos could not distinguish an NT from a control embryo. This unpredictability of individual gene expression on a global background of multiple gene expression changes argues for a predominantly stochastic nature of reprogramming errors.
authors.affiliation: AgResearch Ruakura, Hamilton, New Zealand and University of Waikato, Department of Biological Sciences, Hamilton.
authors.forename: Joanna
authors.initials: J
authors.lastname: Somers
authors.forename: Craig
authors.initials: C
authors.lastname: Smith
authors.forename: Martyn
authors.initials: M
authors.lastname: Donnison
authors.forename: David N
authors.initials: DN
authors.lastname: Wells
authors.forename: Harold
authors.initials: H
authors.lastname: Henderson
authors.forename: Lance
authors.initials: L
authors.lastname: McLeay
authors.forename: P L
authors.initials: PL
authors.lastname: Pfeffer
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D002417:Cattle; D019976:Cloning, Organism; D018076:DNA, Complementary; D046149:Embryo Culture Techniques; D005260:Female; D005307:Fertilization in Vitro; D005347:Fibroblasts / Q000648:ultrastructure*; D015870:Gene Expression; D020869:Gene Expression Profiling; D015723:Gene Library; D006822:Hybrid Cells; D008297:Male; D053652:Nuclear Transfer Techniques; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary