Forty altered genes related to HD from the microarray data.

<p>1094 significant genes were analyzed through the MetaCore built-in HD pathway, and 40 HD related genes were identified. Gene symbols and expression fold-changes are listed.</p><p>Forty altered genes related to HD from the microarray data.</p

Dissection of the methylation status of the putative <i>INS</i> DMR in cloned and wild-type pigs.

<p>(A) A schematic diagram of the pig <i>INS</i> gene showing the relative positions of promoter, exon 1 and CpG islands. The black arrow indicates the location of the primer set. The striped box indicates the putative DMR. The horizontal line indicates the CpG site. The PCR product was digested with <i>Bst</i>UI. (B) The putative DMR of the maternally imprinted <i>INS</i> gene in cloned pigs and wild-type pigs was analyzed by COBRA. The numbers under the figure in the WT panel indicate the average methylation percentage of three wild-type pigs. (C) Bisulfite sequencing of the <i>INS</i> putative DMR in WT ear, CP4 liver, and CP3 ear. Open and closed circles indicate unmethylated and methylated CpG sites, respectively. The top number indicates the CpG site position of the analyzed <i>INS</i> putative DMR. The bottom line indicates the <i>Bst</i>UI recognition site. Fourteen clones of each tissue were sequenced. The methylation percentage calculations were divided into three parts: CpG sites 1 to 20 (all), CpG sites 15 to 20, and CpG sites 13 to 20. For example, WT ear showed a total methylation percentage of 75.7 (212 methylated CpG sites/20 CpG sites ×14 clones = 75.7%). M1: Bio 100 DNA ladder; M2: 500 bp DNA ladder; Br: brain; Ea: ear; He: heart; Ki: kidney; Li: liver; Lu: lung; Mu: muscle; Pl: placenta; Um: umbilical cord; B: blood; S: blood treated with <i>Sss</i>I; W: ddH₂O.</p

Body weight, survival time, and aberrant methylation status of imprinted genes in wild-type and of cloned pigs.

1<p>The body weight of control newborn piglets is an average value from 10 litters (98 piglets) of same-age piglets of the nuclear donor pig breed. WT: wild-type. M: maternally imprinted genes (<i>H19</i> and <i>IGF2R</i>). P: paternally imprinted genes (<i>IGF2</i> and <i>INS</i>).</p

Gene ontology (GO) analysis of microarray in DAVID.

<p>1094 significant genes (<i>P</i> < 0.05) from the microarray were imported into DAVID. GO term analysis was performed to identify biological processes enriched among miR-196a regulated genes (<i>P</i> < 0.05). The top three annotation clusters are shown. Category classifications are based on the KEGG database, which is a collection of biological pathways from “Kyoto Encyclopedia of Genes and Genomes”. DEG: differentially expressed gene.</p><p>Gene ontology (GO) analysis of microarray in DAVID.</p

The aberrant methylation of the <i>H19</i>, <i>INS</i> and <i>IGF2</i> genes and their mRNA expression levels in cloned pigs.

<p>(A) DNA methylation statuses of <i>H19</i>, <i>INS</i>, and <i>IGF2</i> in CP2 ear, CP4 ear, and CP3 ear, respectively. (B) mRNA expression levels of <i>H19</i>, <i>INS</i> and <i>IGF2</i> gene in CP2 ear, CP4 ear, and CP3 ear, respectively. (C) mRNA levels from panel B relative to <i>β-actin</i>.</p

The top five pathways involved in “tissue remodeling and wound repair” via MetaCore analyses.

<p>The orange bar stands for significant genes (<i>P</i> < 0.05) acquired from microarray data, while the blue bar is the expected gene directly interacting with miR-196 from TargetScan. The list of possible affected pathways is arranged as per descending <i>P</i> value score.</p

Identification of the putative DMRs of four imprinted genes and their normal differential methylation patterns in the different tissues of wild-type pigs.

<p>(A) Schematic of CpG site distributions in the putative DMRs of four imprinted genes, <i>IGF2</i>, <i>H19</i>, <i>INS</i>, and <i>IGF2R</i>. Vertical black lines represent each CpG site. Horizontal gray bars represent analyzed regions. Horizontal reticular bars represent putative DMRs in the imprinted genes. Horizontal solid black bars represent probes used for Southern blot hybridization. The upper line indicates the scale bar for DNA length. The putative DMR of <i>H19</i> is located between nt 30,856 and nt 33,489 (GenBank accession no. AY044827). The CpG island of <i>H19</i> corresponds to our designed probe, which ranges from nt 31,411 to nt 31,818. The putative DMR1 of pig <i>IGF2</i> is located between exon 3 and exon 4 and ranges from nt 17,620 to nt 18,796 (GenBank accession no. AY044828). The CpG island of <i>IGF2</i> corresponds to our designed probe, which ranges from nt 17,733 to nt 18,048. The putative DMR2 of <i>IGF2</i> is located in exon 9, nt 27,441 to nt 27,819 (GenBank accession no. AY242102.1). The putative DMR of pig <i>INS</i> is located between nt 1,456 and nt 2,323, and the probe ranges from nt 1,663 to nt 1,986 (GenBank accession no. AY242112). The putative DMR of <i>IGF2R</i> is located between exon 2 and exon 3 (GenBank accession no. AF339885). (B) The normal differential methylation patterns of the four imprinted genes in several tissues of wild-type pigs. The methylation statuses of <i>IGF2</i>, <i>INS</i>, and <i>IGF2R</i> were assayed by COBRA. The methylation status of <i>H19</i> was assayed with Southern blot analysis. The numbers under the images indicate the average methylation percentage in the different tissues of three wild-type pigs (n = 3). Mu: muscle; He: heart; Ea: ear; Li: liver; Lu: lung; Ki: kidney; Br: brain; Pl: placenta.</p

Enrichment analysis by pathway maps in the folder 'Huntington Disease' of MetaCore.

<p>1094 significant genes were analyzed through the MetaCore built-in HD related pathways, and seven critical pathways are listed. “Total” indicates the total number of genes involved in different specific pathways of the MetaCore database. “In Data” indicates the number of significantly altered genes matching the genes in those specific pathways.</p><p>Enrichment analysis by pathway maps in the folder 'Huntington Disease' of MetaCore.</p

Methylation status of the <i>IGF2R</i> putative DMR of intron 2 in cloned pigs and wild-type pigs by COBRA analysis.

<p>(A) Schematic diagram showing the distribution of CpG sites of the pig <i>IGF2R</i> gene. (B) COBRA data showing the methylation status of the <i>IGF2R</i> intron 2 putative DMR in control and wild-type pigs. The numbers under the figure in the control panel indicate the average methylation percentage of three wild-type pigs. Br: brain; Ea: ear; He: heart; Ki: kidney; Li: liver; Lu: lung; Mu: muscle; Pl: placenta; Um: umbilical cord; B: blood; S: blood treated with <i>Sss</i>I; W: ddH₂O.</p

Summary of results from the analyses of different bioinformatic tools.

<p>Microarray data (GSE47500) was used to compare the expression profiling between HD transgenic mice (GHD) and HD transgenic mice overexpressing miR-196a (D-Tg). The potential regulatory pathways are listed via different analyses, including DAVID, MSigDB and MetaCore.</p