|Freking, Bradley - Brad|
|Nonneman, Danny - Dan|
Submitted to: The International Embryo Technology Society
Publication Type: Abstract Only
Publication Acceptance Date: 11/15/2005
Publication Date: 1/8/2006
Citation: Piedrahita, J.A., Bischoff, S.R., Estrada, J.L., Freking, B.A., Nonneman, D.J., Martin, A.C., Mir, B., Rohrer, G.A., Tsai, S. 2006. Use of porcine parthenotes and gene expression profiling using microarrays for identification of imprinted genes [abstract]. Proceedings of the 32nd International Conference of the International Embryo Transfer Society (IETS), January 8-10, 2006, Orlando, FL. Page 239.
Technical Abstract: Genomic imprinting arises from differential epigenetic markings including DNA methylation and histone modifications and results in one allele being expressed in a parent-of-origin specific manner. To gain further insight into the porcine epigenome, gene expression profiles of parthenogenetic (PRT; two maternally-derived chromosome sets; no paternally derived ones) and biparental embryos (BP; one maternal and one paternal set of chromosomes) were compared using pig-specific Affymetrix microarrays. Comparison of the expression profiles of the two tissue types permits identification of both maternally and paternally imprinted genes and thus the degree of conservation of imprinted genes between swine and other mammalian species such as humans and mice. Diploid porcine parthenogenetic fetuses were generated using follicular oocytes (BOMED, Madison, WI). Oocytes containing a visible polar body were activated using a single square pulse of direct current of 50 V/mm for 100 u-sec and diploidized by culture in 10 ug/mL cycloheximide for 6 h to limit extrusion of the second polar body. Following culture, BP embryos obtained by natural matings and PRT embryos were surgically transferred to gilt oviducts on the first day of estrus. Fetuses recovered at 28-30 days of gestation were mechanically dissected to separate viscera including brain, liver, and placenta, then flash-frozen in liquid nitrogen. Porcine fibroblast tissue was obtained from the remaining carcass by mincing, trypsinization, and plating cells in MEM. Total RNA was extracted from frozen tissue or cell culture using RNA Aqueous kit (Ambion, Austin, TX) according to the manufacturer’s protocol. Gene expression differences between SP and PRT tissues were determined using the GeneChip® Porcine Genome Array (Affymetrix, Santa Clara, CA) containing 23,256 transcripts from Sus scrofa and representing 42 genes known to be imprinted genes in human and/or mice. Triplicate arrays were utilized for each tissue type, and PRT versus BP combination. Data was analyzed using a mixed-model approach in conjunction with SAS Microarray Solutions software. False-discovery-rate multiple correction testing was performed on each comparison and significance was set at (Q < 0.05). The following genes were determined to be imprinted: In fibroblasts, ARHI, HTR2A, MEST, NDN, NNAT, PEG3, PLAGL1, PEG10, SGCE, SNRPN, and UBE3A. In liver, IGF2, PEG3, PLAGGL1, PEG10, and SNRPN. In placenta, HTR2A, IGF2, MEST, NDN, NNAT, PEG3, PLAGL1, PEG10, and SNRPN. And in brain, none. Additionally, several genes not known to be imprinted in humans/mice were highly differentially expressed between the two tissue types. Overall, where imprinting is conserved between swine and humans/mice, and several candidate genes that represent potentially imprinted genes. Presently, our efforts are focused in the identification of single nucleotide polymorphisms (SNP’s) to more carefully evaluate the behavior of these genes in normal and abnormal gestations and to test whether the candidate genes are indeed imprinted. This research was supported by USDA grant 524383 to JP and BF.