Genetic association of polymorphisms in porcine RGS16 with porcine circovirus viral load in naturally infected Yorkshire pigs.

Regulator of G protein signaling 16 (RGS16) is known to be associated with porcine circovirus type 2 (PCV2). PCV2 associated disease (PCVAD) is a serious problem in the swine industry. The representative symptoms of PCVAD are high viral titer proliferation and decreased average daily gain. In this study, we identified single nucleotide polymorphisms (SNPs) in the RGS16 region, including the upstream region. Of the 22 identified SNPs, rs332913874, rs326071195, and rs318298586 were genotyped in 142 Yorkshire pigs. These SNPs were significantly associated with the PCV2 viral load. Moreover, the haplotype combination was also related to the PCV2 viral load. The haplotype and diplotype analysis also had a significant difference with the PCV2 viral load. Taken together, our results suggest that RGS16 SNPs considerably affect the PCV2 viral load.

Evaluation of whole pork belly qualitative and quantitative properties using selective belly muscle parameters.

The objective of this study was to identify parameters for the evaluation of pork belly quality (composition) and quantity (volume) and to develop regression equations that predict properties of whole pork belly. Through an image analysis of 648 bellies, newly characterized pork belly parameters were developed for evaluating pork belly quality and quantity. Importantly, the estimated muscle volume showed high positive correlation with the whole belly volume and the whole belly muscle percentage (r=0.458, and 0.654, respectively). Section 7 was identified as the best section for the evaluation of pork belly based on the muscle area in every vertebra. A stepwise regression showed that cutaneous trunci muscle (CTM) had an r2 of 0.624 in the model, and supplementation with the other muscles yielded an r2 of 0.784. Therefore, we propose that a prediction equation could be developed for a certain area in the belly for the evaluation of pork belly quantity and quality. The results could be applied to select breeding stock using techniques such as ultrasound with the aim of producing hogs with large as well as lean bellies.

Genomic structure, expression and association study of the porcine FSD2.

The fibronectin type III and SPRY domain containing 2 (FSD2) on porcine chromosome 7 is considered a candidate gene for pork quality, since its two domains, which were present in fibronectin and ryanodine receptor. The fibronectin type III and SPRY domains were first identified in fibronectin and ryanodine receptor, respectively, which are candidate genes for meat quality. The aim of this study was to elucidate the genomic structure of FSD2 and functions of single nucleotide polymorphisms (SNPs) within FSD2 that are related to meat quality in pigs. Using a bacterial artificial chromosome clone sequence, we revealed that porcine FSD2 consisted of 13 exons encoding 750 amino acids. In addition, FSD2 was expressed in heart, longissimus dorsi muscle, psoas muscle, and tendon among 23 kinds of porcine tissues tested. A total of ten SNPs, including four missense mutations, were identified in the exonic region of FSD2, and two major haplotypes were obtained based on the SNP genotypes of 633 Berkshire pigs. Both haplotypes were associated significantly with intramuscular fat content (IMF, P < 0.020) and moisture percentage (MP, P < 0.002). Moreover, haplotype 2 was associated with meat color, affecting yellowness (P = 0.002). These haplotype effects were further supported by the alteration of putative protein structures with amino acid substitutions. Taken together, our results suggest that FSD2 haplotypes are involved in regulating meat quality including IMF, MP, and meat color in pigs, and may be used as meaningful molecular makers to identify pigs with preferable pork quality.

Association of a single nucleotide polymorphism in the 5' upstream region of the porcine myosin heavy chain 4 gene with meat quality traits in pigs.

We identified a potential molecular marker associated with meat quality traits in the myosin heavy chain 4, MYH4 gene of Landrace pigs. Sequencing revealed a single nucleotide polymorphism (SNP; g.-1398G>T) in the 5' upstream region of MYH4. It was significantly associated with the number of type IIa muscle fibers and water-holding capacity based on filter-paper fluid uptake. The GG genotype groups had a greater number of type IIa fibers and a larger area composed of type IIa fibers than the other genotype group (P = 0.004 and P = 0.061, respectively). Expression level of MYH4 gene in the genotype TT or GT was higher than in genotype of GG (P < 0.0001). The T allele may enhance expression level of MYH4 gene and then the portion of IIb type fiber in the muscle be increased by the T allelle. Therefore, we suggest that the g.-1398G>T in the 5' upstream region of the porcine MYH4 may be used as a molecular marker for meat quality traits, although its functional effect is not defined yet.

Effects of intergenic single nucleotide polymorphisms in the fast myosin heavy chain cluster on muscle fiber characteristics and meat quality in Berkshire pigs.

The heterogeneity of myosin heavy chain (MyHC) isoforms is closely related to muscle fiber characteristics, and meat quality in pigs. The fast MyHC gene cluster on porcine chromosome 12 contains MYH2, MYH1, and MYH4, which encode three fast MyHC isoforms expressed in adult skeletal muscle. Here, we identified four intergenic single nucleotide polymorphisms (SNPs) in the gene cluster. Of these, SNP1 and SNP2, which were located near MYH2, were genotyped in 199 Berkshire pigs. SNP1 were significantly associated with the total fiber number (P=0.046) and intramuscular fat contents (P=0.041), and SNP2 had significant effects on type I fiber number/area compositions (P=0.039 and P=0.041, respectively), water holding capacity (drip loss, P=0.045; cooking loss, P=0.001), and meat color (P=0.003). Taken together, our results suggest that the intergenic SNPs near MYH2 can affect muscle fiber formation and meat quality by modifying the regulatory elements of this gene cluster in pigs.

A Candidate Single Nucleotide Polymorphism in the 3' Untranslated Region of Stearoyl-CoA Desaturase Gene for Fatness Quality and the Gene Expression in Berkshire Pigs.

Fatness qualities in pigs measured by the amount of fat deposition and composition of fatty acids (FAs) in pork have considerable effect on current breeding goals. The stearoyl-CoA desaturase (SCD) gene plays a crucial role in the conversion of saturated FAs into monounsaturated FAs (MUFAs), and hence, is among the candidate genes responsible for pig fatness traits. Here, we identified a single nucleotide polymorphism (SNP, c.*2041T>C) in the 3' untranslated region by direct sequencing focused on coding and regulatory regions of porcine SCD. According to the association analysis using a hundred of Berkshire pigs, the SNP was significantly associated with FA composition (MUFAs and polyunsaturated FAs [PUFAs]), polyunsaturated to saturated (P:S) FA ratio, n-6:n-3 FA ratio, and extent of fat deposition such as intramuscular fat and marbling (p<0.05). In addition, the SNP showed a significant effect on the SCD mRNA expression levels (p = 0.041). Based on our results, we suggest that the SCD c.*2041T>C SNP plays a role in the gene regulation and affects the fatness qualities in Berkshire pigs.

Effects of polymorphisms in the porcine microRNA MIR206 / MIR133B cluster on muscle fiber and meat quality traits.

MicroRNAs are a class of small non-coding RNA molecules that repress gene expression primarily at the post-transcriptional level. Genetic variations in microRNA genes may contribute to phenotypic differences by altering the expression of microRNAs and their targets. Here, we identified 12 single nucleotide polymorphisms (SNPs) in the genomic region of the porcine MIR206 / MIR133B cluster, 10 and 2 of which were associated with MIR206 and MIR133B respectively. All 12 SNPs were located within primary microRNAs. Allele frequency determination in different pig breeds (Berkshire, n = 153; Landrace, n = 125; Yorkshire, n = 173) and association studies of muscle fiber characteristics, lean meat production and meat quality traits were performed on the MIR206 and MIR133B SNPs. The MIR206 SNPs were associated with the percentage of type IIa and IIb fibers for muscle fiber area composition, meat quality traits including drip loss and lightness, and backfat thickness, a parameter of lean meat production. In addition, we found significant association of the MIR133B SNPs with total muscle fiber number, loin eye area, and muscle pH. Furthermore, these SNPs significantly affected the levels of mature MIR206 and MIR133B , respectively, primarily by regulating the processing of primary microRNAs into precursor microRNAs. Interestingly, altered MIR206 levels correlated with phenotypic variability among genotypes of the MIR206 SNP. Our data suggest that polymorphisms in the porcine MIR206 / MIR133B cluster are a genetic factor affecting muscle and meat quality traits.

Effects of polymorphisms in the porcine microRNA miR-1 locus on muscle fiber type composition and miR-1 expression.

MicroRNAs (miRNAs) are a group of evolutionarily conserved small noncoding RNAs with regulatory functions. Increasing evidence suggests that polymorphisms in miRNA genes are associated with phenotypic variation by affecting miRNA expression and/or function. Here, we identified two single nucleotide polymorphisms (SNPs) in the porcine miR-1 locus, both of which were linked and located downstream from the stem-loop miRNA precursor sequence within the primary miR-1 region. An association study on muscle fiber characteristics and meat quality traits was performed with a total of 451 pigs representing three pig breeds (Berkshire, n=153; Landrace, n=125; Yorkshire, n=173). The miR-1 SNPs were significantly associated with type I and type IIa muscle fibers in number and area compositions, respectively, but not with meat quality traits. Notably, these polymorphisms were also significantly associated with altered expression of the primary miR-1 transcript, ultimately leading to comparable changes in the levels of both precursor and mature miR-1. Furthermore, altered miR-1 levels were correlated with the variation in muscle fiber composition. Our data suggest that miR-1 may be a candidate gene associated with muscle fiber type composition.

Effects of polymorphisms in the 3' untranslated region of the porcine PPARGC1A gene on muscle fiber characteristics and meat quality traits.

Peroxisome proliferator-activated receptor γ coactivator 1 α (PPARGC1A) is a transcriptional coactivator that is involved in a variety of biological processes including muscle fiber type composition. Here, we identified two single nucleotide polymorphisms (SNPs; *2690T>C and *2864T>C) and one insertion/deletion in the 3' untranslated region of porcine PPARGC1A. These SNPs were genotyped by direct sequencing in a total of 439 pigs representing three different pig breeds (Berkshire, n = 156; Yorkshire, n = 163; Landrace, n = 120). We evaluated the effects of diplotypes of individual PPARGC1A 3'UTR SNPs on muscle fiber characteristics and meat quality traits. The *2690T>C polymorphism was significantly associated with the percentage of type I and IIb fibers for both muscle fiber number and area composition (P < 0.05), and also showed a significant association with muscle pH, a parameter of meat quality (P = 0.0188). The *2864T>C polymorphism was also associated with meat quality traits including muscle pH (P = 0.0071), drip loss (P = 0.0006), and lightness (P = 0.0702), but showed no significant association with muscle fiber characteristics. Interestingly, each SNP affected PPARGC1A expression significantly at the protein level but not at the mRNA level, thereby accounting for phenotypic variability among genotypes. Taken together, our data suggest that the *2690T>C and *2864T>C polymorphisms can be used as genetic markers for selection toward improved meat quality.

Cloning and characterization of microRNAs from porcine skeletal muscle and adipose tissue.

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.

WDNM1 is associated with differentiation and apoptosis of mammary epithelial cells.

In this study, we show that expression of the Westmead DMBA8 nonmetastatic cDNA 1 (WDNM1) gene was increased upon SFM and/or TNFalpha treatment, with a corresponding increase in apoptotic cells, and gradually decreased following re-stimulation with serum in HC11 mammary epithelial cells. TNFalpha induced WDNM1 expression showed the NFkappaB-dependent mechanism since it's expression was abrogated in IkappaBalphaM (super-repressor of NFkappaB)-transfected cells, but not those transfected with control vector. Furthermore, overexpression of WDNM1 suppressed growth and differentiation, and accelerated apoptosis of HC11 cells. Thus, our results demonstrate that WDNM1 gene expression, regulated by the TNFalpha-NFkappaB signal pathway, is associated with HC11 cell apoptosis.

Induction of serum amyloid A genes is associated with growth and apoptosis of HC11 mammary epithelial cells.

In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFalpha treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IkappaBalpha. Similar results were observed in response to interleukin-1 (IL-1), IL-6 and interferon gamma (IFNgamma). Furthermore, overexpression of the SAA1 and SAA2 isoforms suppressed growth and accelerated apoptosis of HC11 cells by increasing caspase 3/7 and caspase 8 activities, but the apoptotic effect of tumor necrosis factor alpha (TNFalpha) on HC11 cells was not enhanced. We found that expression of SAA1 and SAA2, but not SAA3, was regulated by an NFkappaB-dependent pathway, and that overexpression of SAA isoforms accelerated the apoptosis of HC11 cells.

Characteristics of primary and immortalized fibroblast cells derived from the miniature and domestic pigs.

BACKGROUND: The pig, Sus scrofa domestica includes both the miniature and commercial domestic breed. These animals have influenced the human life and economies and have been studied throughout history. Although the miniature breeds are more recent and have increasingly been used in a variety of biomedical studies, their cell lines have rarely been established. Therefore, we sought to establish primary and immortal cell lines derived from both the miniature and domestic pig to better enable insight into possible in vivo growth differences. RESULTS: The in vitro lifespan of primary domestic pig fibroblast (PF) and miniature pig fibroblast (MPF) cells using a standard 3T3 protocol was determined. Both of the primary PF and MPF cells were shown to have a two-step replicative senescence barrier. Primary MPF cells exhibited a relatively shorter lifespan and slower proliferation rate compared to those of primary PF cells. Beyond senescence barriers, lifespan-extended PF and MPF cells were eventually established and indicated spontaneous cellular immortalization. In contrast to the immortalized PF cells, immortal MPF cells showed a transformed phenotype and possessed more frequent chromosomal abnormalities and loss of p53 regulatory function. The lifespan of primary MPF and PF cells was extended by inactivation of the p53 function using transduction by SV40LT without any detectable senescent phenotype. CONCLUSION: These results suggest that p53 signaling might be a major determinant for the replicative senescence in the MPF cells that have the shorter lifespan and slower growth rate compared to PF cells in vitro.

Myogenic differentiation of p53- and Rb-deficient immortalized and transformed bovine fibroblasts in response to MyoD.

We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and p16(INK4a) functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and p16(INK4a) pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.

Activation of the intrinsic mitochondrial apoptotic pathway in swine influenza virus-mediated cell death.

The mitochondrial pathway of swine influenza virus (SIV)-induced apoptosis was investigated using porcine kidney (PK-15) cells, swine testicle (ST) cells, and HeLa cervical carcinoma cells which are known not to support viral replication. As judged by cell morphology, annexin V staining, and DNA fragmentation, PK-15 and ST cells infected with three different subtypes of SIV (H1N1, H3N2, and H1N2) were obviously killed by apoptosis, not necrosis. SIV infection in PK-15 and HeLa cells was shown to decrease the cellular levels of Bcl-2 protein compared to that of mock-infected control cells at 24 h post-infection, whereas expression levels of Bax protein increased in the PK-15 cells, but did not increase in HeLa cells by SIV infection. Cytochrome c upregulation was also observed in cytosolic fractions of the PK-15 and HeLa cells infected with SIV. Apoptosome (a multi-protein complex consisting of cytochrome c, Apaf-1, caspase-9, and ATP) formation was confirmed by immunoprecipitation using cytochrome c antibody. Furthermore, SIV infection increased the cellular levels of TAJ, an activator of the JNK- stressing pathway, and the c-Jun protein in the PK-15 and HeLa cells. Taken together, these results suggest that the mitochondrial pathway should be implicated in the apoptosis of PK-15 cells induced by SIV infection.

Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells.

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.

Cellular characteristics of primary and immortal canine embryonic fibroblast cells.

Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.