DIFFERENTIAL GENE EXPRESSION PROFILING OF DAY 45 PLACENTA IN CHROMATIN TRANSFER AND IN VITRO FERTILIZATION-DERIVED CATTLE FETUSES EVALUATED BY TWO MICROARRAY PLATFORMS

Abstract

Nuclear transfer has been used to produce genetically identical farm animals for a variety of purposes. Recently, an alternative cloning method called chromatin transfer (CT) was developed to produce cloned cattle. While the success rate of CT is higher than for nuclear transfer it is still far from optimal and is also likely caused by abnormal expression of specific genes. A common abnormality seen in somatic cell-derived pregnancies is Large Offspring Syndrome (LOS). This syndrome involves developmental abnormalities not only in the fetus but also in the placenta. The present study was designed to compare differences in patterns of gene expression between placentas obtained from CT and IVF fetuses using two different microarray platforms. We hypothesized that the abnormal placentation process associated with LOS is due to aberrant gene expression patterns. Two different microarray technologies were used.. Complementary DNA (cDNA) was reverse-transcribed from total RNA of day 45 placentas from two CT and two IVF bovine fetuses and hybridized to two different microarrays. The first was the Pyxis microarray (Pyxis Genomics) which was generated from normalized bovine spleen and placental cDNA libraries containing nearly 7600 double-spotted genes and the second was the Affymetrix GeneChip oligonucleotide array containing more than 23,000 bovine transcripts. The hybridization intensities were Log2-transformed and analyzed by an unpaired t-Test parametric model. Changes in gene expression induced by the treatment (CT) were considered significant if a gene had a p-value < 0.05 and a ±1.1 fold change compared to IVF. A total of 769 genes showed differential gene expression profiles on the affymetrix chip, while the Pyxis array had 233 differentially expressed genes. Cross comparison between the two arrays identified 50 genes showing the same change in expression pattern on the two arrays. These included 11 ESTs, 8 enzymes, 7 adhesion molecules, 5 oncogenes, 12 cell growth 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 genes significantly up or down regulated on the microarray platforms. The criteria used to select the genes from the microarrays were based on biological and statistical significance. Out of 10 genes tested, TIMP-3 and TKDP4 were confirmed as up-regulated in the CT samples while p57 and LAMA3 were confirmed to be down-regulated by qRT-PCR. An additional 4 IVF samples and 4 CT samples were analyzed on the Affymetrix array and the same 4 genes were confirmed to be different in these samples. Despite the relatively low number of samples analyzed on the microarrays we were able to identify genes that showed consistent differences in pattern of expression on both arrays. There was a significant number of genes that did not show the same trends on the two microarrays. Future studies will focus on determining how altered expression of these four genes may contribute to the abnormal placentation observed in LOS. (platform)