Gene expression profiling of individual bovine nuclear transfer blastocysts.
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.
Somers
Joanna
J
AgResearch Ruakura, Hamilton, New Zealand and University of Waikato, Department of Biological Sciences, Hamilton.
Smith
Craig
C
Donnison
Martyn
M
Wells
David N
DN
Henderson
Harold
H
McLeay
Lance
L
Pfeffer
P L
PL
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
D018076:DNA, Complementary