Shengdar Tsai

Characterization of Conserved and Nonconserved Imprinted Genes in Swine

To increase our understanding of imprinted genes in swine, we carried out a comprehensive analysis of this gene family using two complementary approaches: expression and phenotypic profiling of parthenogenetic fetuses, and analysis of imprinting by pyrosequencing. The parthenote placenta and fetus were smaller than those of controls but had no obvious morphological differences at Day 28 of gestation. By Day 30, however, the parthenote placentas had decreased chorioallantoic folding, decreased chorionic ruggae, and reduction of fetal-maternal interface surface in comparison with stage-matched control fetuses. Using Affymetrix Porcine GeneChip microarrays and/or semiquantitative PCR, brain, fibroblast, liver, and placenta of Day 30 fetuses were profiled, and 25 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. Samples were collected from Day 30 pregnancies generated from reciprocal crosses of Meishan and White Composite breeds, and single-nucleotide polymorphisms were identified in candidate genes. Imprinting was confirmed 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. Combined, these results represent the most comprehensive survey of imprinted genes in swine to date.

Transcriptional profiling of human placentas from pregnancies complicated by preeclampsia reveals disregulation of sialic acid acetylesterase and immune signalling pathways.

The placenta plays an important role as a regulator of fetal nutrition and growth throughout development and placental factors contribute to gestational abnormalities such as preeclampsia. This study describes the genome-wide gene expression profiles of a large (n = 60) set of human placentas in order to uncover gene expression patterns associated with preeclampsia. In addition to confirming changes in expression of soluble factors associated with preeclampsia such as sFLT1 (soluble fms-like tyrosine kinase-1), sENG (soluble endoglin), and INHA (inhibin alpha), we also find changes in immune-associated signaling pathways, offering a potential upstream explanation for the shallow trophoblast invasion and inadequate uterine remodeling typically observed in pathogenesis of preeclampsia. Notably, we also find evidence of preeclampsia-associated placental upregulation of sialic acid acetylesterase (SIAE), a gene functionally associated with autoimmune diseases.

Detection of transcriptional difference of porcine imprinted genes using different microarray platforms

BackgroundPresently, multiple options exist for conducting gene expression profiling studies in swine. In order to determine the performance of some of the existing microarrays, Affymetrix Porcine, Affymetrix Human U133+2.0, and the U.S. Pig Genome Coordination Program spotted glass oligonucleotide microarrays were compared for their reproducibility, coverage, platform independent and dependent sensitivity using fibroblast cell lines derived from control and parthenogenic porcine embryos.ResultsArray group correlations between technical replicates demonstrated comparable reproducibility in both Affymetrix arrays. Glass oligonucleotide arrays showed greater variability and, in addition, approximately 10% of probes had to be discarded due to slide printing defects. Probe level analysis of Affymetrix Human arrays revealed significant variability within probe sets due to the effects of cross-species hybridization. Affymetrix Porcine arrays identified the greatest number of differentially expressed genes amongst probes common to all arrays, a measure of platform sensitivity. Affymetrix Porcine arrays also identified the greatest number of differentially expressed known imprinted genes using all probes on each array, an ad hoc measure of realistic performance for this particular experiment.ConclusionWe conclude that of the platforms currently available and tested, the Affymetrix Porcine array is the most sensitive and reproducible microarray for swine genomic studies.

Growth requirements and chromosomal instability of induced pluripotent stem cells generated from adult canine fibroblasts.

In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors, Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Here, we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [POU5F1 (OCT4), SOX2, NANOG, and LIN28] and embryonic stem cell (ESC)-specific genes (PODXL, DPPA5, FGF5, REX1, and LAMP1) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo, the ciPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown.

Successful Cloning of the Yucatan Minipig Using Commercial/Occidental Breeds as Oocyte Donors and Embryo Recipients

The widespread application of porcine SCNT to biomedical research is being hampered by the large adult size (300-600 lbs) of the commercial breeds commonly used for SCNT. The Yucatan minipig, in contrast, has an adult weight of 140-150 lbs and a long history of utility in biomedical research. In order to combine the wide availability of commercial swine with the biomedical value of the Yucatan minipig, we utilized SCNT using the Yucatan as nuclear donors and commercial swine as both oocyte donors and recipients. Of six recipient gilts receiving 631 SCNT embryos, three went to term and delivered seven piglets, four of which survived to adulthood. Additionally, we obtained fetal fibroblasts from a cloned Yucatan and used them for a second round of SCNT. Of three recipients receiving 315 reconstructed embryos, one went to term and delivered three piglets, one of which survived to adulthood. Both microsatellite and D-loop sequence analysis confirmed that all of the piglets generated were nuclear-mitochondrial hybrids carrying Yucatan nuclear DNA and commercial breed mitochondrial DNA. This report shows that it is possible to produce viable Yucatan SCNT clones and opens up the possibility of developing valuable biomedical models in this porcine breed.

Identification of SNPs and INDELS in swine transcribed sequences using short oligonucleotide microarrays

BackgroundGenome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to identify significant breed-by-probe interactions.ResultsGene specific linear mixed models were fit to each of the log2 transformed probe intensities on these arrays, using fixed effects for breed, probe, breed-by-probe interaction, and a random effect for array. After surveying the day 25 placental transcriptome, 857 probes with a q-value ≤ 0.05 and |fold change| ≥ 2 for the breed-by-probe interaction were identified as candidates containing SFP. To address the quality of the bioinformatics approach, universal pyrosequencing assays were designed from Affymetrix exemplar sequences to independently assess polymorphisms within a subset of probes for validation. Additionally probes were randomly selected for sequencing to determine an unbiased confirmation rate. In most cases, the 25-mer probe sequence printed on the microarray diverged from Meishan, not occidental crosses. This analysis was used to define a set of highly reliable predicted SFPs according to their probability scores.ConclusionBy applying a SFP detection method to two mammalian breeds for the first time, we detected transition and transversion single nucleotide polymorphisms, as well as insertions/deletions which can be used to rapidly develop markers for genetic mapping and association analysis in species where high density genotyping platforms are otherwise unavailable.SNPs and INDELS discovered by this approach have been publicly deposited in NCBIs SNP repository dbSNP. This method is an attractive bioinformatics tool for uncovering breed-by-probe interactions, for rapidly identifying expressed SNPs, for investigating potential functional correlations between gene expression and breed polymorphisms, and is robust enough to be used on any Affymetrix gene expression platform.

Lack of genomic imprinting of DNA primase, polypeptide 2 (PRIM2) in human term placenta and white blood cells.

PRIM2, encoding a subunit of primase involved in DNA replication and transcription, is expressed in the placenta and is crucial for mammalian development and growth. Its role in placental function is not well understood. Recently, PRIM2 was reported as imprinted in human white blood cells (WBC). We report here our failure to confirm imprinting of the PRIM2 locus in human placenta or WBC. The discordance between our results and those of others are likely due to an incorrectly annotated PRIM2 pseudogene found in the human genome database.