January 9-13, 2010
Town & Country Convention Center
San Diego, CA
Brad A. Freking1 , Steve R. Bischoff2 , Shengdar Tsai2 , Nicholas Hardison2 , Alison A. Motsinger-Reif2 , Dan Nonneman1 , Gary Rohrer1 , Jorge A. Piedrahita2
Genomic imprinting results in the silencing of a subset of mammalian alleles due to parent-of-origin inheritance. Due to the nature of their expression patterns they play a critical role in placental and early embryonic development. In order to increase our understanding of imprinted genes specifically in swine, we carried out a comprehensive analysis of this set of genes using multiple complementary approaches, expression and phenotypic profiling of parthenogenetic fetuses, and analysis of imprinting by pyrosequencing and MALDI-TOF genotyping. Utilizing Affymetrix Porcine GeneChip microarrays and/or semi-quantitative PCR, brain, fibroblast, liver, and placenta of day 30 fetuses were profiled and twenty-five imprinted genes were identified as differentially expressed in at least one of the four tissue types: AMPD3, CDKN1C, COPG2, DHCR7, DIRAS3, IGF2 (isoform specific), IGF2AS, IGF2R, MEG3, MEST, NAP1L5, NDN, NNAT, OSBPL1A, PEG3, APEG3, PEG10, PLAGL1, PON2, PPP1R9A, SGCE, SLC38A4, SNORD107, SNRPN, and TFPI2. For DIRAS3, PLAGL1, SGCE and SLC38A4 tissue-specific differences were detected. In addition, we examined the imprinting status of candidate genes by quantitative allelic pyrosequencing and by MALDI-TOF genotyping. Samples were collected from day 30 pregnancies generated from reciprocal crosses of Meishan and White Composite breeds, and SNPs identified in candidate genes. Imprinting was confirmed by pyrosequencing for DIRAS3, DLK1, H19, IGF2AS, NNAT, MEST, PEG10, PHLDA2, PLAGL1, SGCE, and SNORD107. We also found no evidence of imprinting in ASB4, ASCL2, CD81, COMMD1, DCN, DLX5, and H13. Twenty additional novel loci had variable levels of detectable allelic suppression by genotyping genomic and embryonic cDNA templates for multiple SNP. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date.
This project was supported by National Research Initiative Grant no. 2005-35604-15343 from the USDA Cooperative State Research, Education, and Extension Service to JP and BF.