Differential Gene Expression Profiling of Day 45 Placenta in Chromatin Transfer and in Vitro Fertilization-Derived Cattle Fetuses Evaluated by Two Microarray Platforms
40th Annual Meeting of the Society for the Study of Reproduction. San Antonio, Texas, USA, July 21–25, 2007
Nuclear transfer has been used to produce genetically identicalfarm animals for a variety of purposes. Recently, an alternativecloning method called chromatin transfer (CT) was developedto produce cloned cattle. While the success rate of CT is higherthan for nuclear transfer it is still far from optimal and isalso likely caused by abnormal expression of specific genes.A common abnormality seen in somatic cell-derived pregnanciesis Large Offspring Syndrome (LOS). This syndrome involves developmentalabnormalities not only in the fetus but also in the placenta.The present study was designed to compare differences in patternsof gene expression between placentas obtained from CT and IVFfetuses using two different microarray platforms. We hypothesizedthat the abnormal placentation process associated with LOS isdue to aberrant gene expression patterns. Two different microarraytechnologies were used.. Complementary DNA (cDNA) was reverse-transcribedfrom total RNA of day 45 placentas from two CT and two IVF bovinefetuses and hybridized to two different microarrays. The firstwas the Pyxis microarray (Pyxis Genomics) which was generatedfrom normalized bovine spleen and placental cDNA libraries containingnearly 7600 double-spotted genes and the second was the AffymetrixGeneChip oligonucleotide array containing more than 23,000 bovinetranscripts. The hybridization intensities were Log2-transformedand analyzed by an unpaired t-Test parametric model. Changesin gene expression induced by the treatment (CT) were consideredsignificant if a gene had a p-value < 0.05 and a ±1.1fold change compared to IVF. A total of 769 genes showed differentialgene expression profiles on the affymetrix chip, while the Pyxisarray had 233 differentially expressed genes. Cross comparisonbetween the two arrays identified 50 genes showing the samechange in expression pattern on the two arrays. These included11 ESTs, 8 enzymes, 7 adhesion molecules, 5 oncogenes, 12 cellgrowth and proliferation proteins, 5 cell trafficking molecules,5 signaling transduction molecules and 5 transcription factors.Quantitative real time PCR (qRT-PCR) was used to validate genessignificantly up or down regulated on the microarray platforms.The criteria used to select the genes from the microarrays werebased on biological and statistical significance. Out of 10genes tested, TIMP-3 and TKDP4 were confirmed as up-regulatedin the CT samples while p57 and LAMA3 were confirmed to be down-regulatedby qRT-PCR. An additional 4 IVF samples and 4 CT samples wereanalyzed on the Affymetrix array and the same 4 genes were confirmedto be different in these samples. Despite the relatively lownumber of samples analyzed on the microarrays we were able toidentify genes that showed consistent differences in patternof expression on both arrays. There was a significant numberof genes that did not show the same trends on the two microarrays.Future studies will focus on determining how altered expressionof these four genes may contribute to the abnormal placentationobserved in LOS. (platform)
Machado S., Mesquita F., Edwards A, Wang Z., P. Kasinathan, Robl J., and Nowak R. Differential gene expression profiling of day 45 placenta in chromatin transfer and in vitro fertilization-derived cattle fetuses evaluated by two microarray platforms. 40th Annual Meeting of the Society for the Study of Reproduction. Abstract. San Antonio, Texas, USA, July 21–25, 2007.