Biomedical subjects

Siriluck Ponsuksili

Publications and source records attributed to Siriluck Ponsuksili.

15 recordsLinked to original sources

PubMed2006

QTL for microstructural and biophysical muscle properties and body composition in pigs.

BACKGROUND: The proportion of muscle fibre types and their size affect muscularity as well as functional properties of the musculature and meat quality. We aimed to identify QTL for microstructural muscle properties including muscle fibre size, their numbers and fibre type proportions as well as biophysical parameters of meat quality and traits related to body composition, i.e. pH, conductivity, area of M. longissimus dorsi and lean meat content. A QTL scan was conducted in a porcine experimental population that is based on Duroc and Berlin Miniature Pig. RESULTS: Least square regression interval mapping revealed five significant and 42 suggestive QTL for traits related to muscle fibre composition under the line-cross model as well as eight significant and 40 suggestive QTL under the half-sib model. For traits related to body composition and biophysical parameters of meat quality five and twelve significant plus nine and 22 suggestive QTL were found under the line-cross and half-sib model, respectively. Regions with either significant QTL for muscle fibre traits or significant QTL for meat quality and muscularity or both were detected on SSC1, 2, 3, 4, 5, 13, 14, 15, and 16. QTL for microstructural properties explained a larger proportion of variance than did QTL for meat quality and body composition. CONCLUSION: Microstructural properties of pig muscle and meat quality are governed by genetic variation at many loci distributed throughout the genome. QTL analysis under both, the line-cross and half-sib model, allows detecting QTL in case of fixation or segregation of the QTL alleles among the founder populations and thus provide comprehensive insight into the genetic variation of the traits under investigation. Genomic regions affecting complex traits of muscularity and meat quality as well as microstructural properties might point to QTL that in first instance affect muscle fibre traits and by this in second instance meat quality. Disentangling complex traits in their constituent phenotypes might facilitate the identification of QTL and the elucidation of the pleiotropic nature of QTL effects.

Animals↗

PubMed2006

Molecular characterization and evidencing of the porcine CRH gene as a functional-positional candidate for growth and body composition.

Corticotropin-releasing hormone (CRH), a major regulator of neuroendocrine response to stress, is involved in the control of energy balance and thus may affect body composition and growth. The porcine CRH (pCRH) gene was studied as a comparative-positional candidate for QTL for longissimus muscle area, average backfat thickness, carcass length, and average daily gain on test on porcine chromosome 4. Sequence of the complete transcriptional unit of pCRH gene spanning 2068bp was determined along with 582bp of the 5'-flanking region. Cross-species sequence comparison revealed a number of potential regulatory regions including an intronic evolutionary conserved region and an adjacent CpG island that may control cell-type specific expression of the CRH gene. A SNP in exon 2 (c.+83G>A) leading to a non-conservative amino acid exchange (p.28Arg>Gln) in the prohormone was identified that is segregating in the DUMI resource population. Linkage and association analysis based on this SNP revealed that for all four traits the pCRH gene falls in the QTL peak area and that the c.+83G>A SNP shows a highly significant additive effect (p<0.0001). Physical mapping using the IMpRH panel assigned the pCRH gene to interval SW724-S0107, promoting the gene as a positional candidate also for QTL identified in other porcine resource populations. Additional four variable sites were identified that segregate in commercial pig breeds. Particularly interesting is a SNP (g.233C>T) in the 5'-flanking region that occurred in an evolutionary conserved motif. The knowledge of the DNA-variation of pCRH gene will facilitate follow-up studies necessary to provide definite genetic evidence of the effect of pCRH gene on body composition and growth.

Amino Acid Sequence↗

PubMed2005

Bovine NALP5, NALP8, and NALP9 genes: assignment to a QTL region and the expression in adult tissues, oocytes, and preimplantation embryos.

A 3204-bp full-length cDNA of bovine NALP9 was cloned and its genomic organization was analyzed. The 2988-bp open reading frame covers 9 exons and encodes a deduced protein of 996 amino acids containing Pyrin, Nacht and leucine-rich repeat domains like the human NALP gene family members. Mapping with the WGRH5000 panel and fluorescence in situ hybridization assigned NALP9 in close vicinity to BM2078 (LOD score 25.71; distance 0.0 cR5000) on bovine chromosome 18, BTA18q25-q26, within a previously identified QTL region for reproductive traits flanked by the bovine marker BM2078 and TGLA227. BAC contig analysis revealed that NALP9, NALP8, and NALP5 map in this QTL region. Temporospatial expression of these members of the NALP gene family was monitored. Among the adult tissues examined, transcripts of NALP8 and NALP9 were detected exclusively in testis and ovary, whereas transcripts of the NALP5 gene are limited to the ovary. The transcripts of NALP9, NALP8, and NALP5 were detected in oocytes before and after in vitro maturation and with a gradual decline from 2-cell to 8-cell stage, suggesting no reactivation at the time of bovine maternal to embryonic transition. Assignment to a QTL region for reproductive traits and preferential expression of NALP9, NALP8, and NALP5 in oocyte, germinal lineage, and gonad cells may suggest their functional relevance to reproduction and possible contribution to phenotypic variation.

Age Factors↗

PubMed2005

Identification of functional candidate genes for body composition by expression analyses and evidencing impact by association analysis and mapping.

This study aims to identify hepatic genes affecting traits related to muscularity and obesity by combining expression analyses, association studies, and gene mapping. Functional candidate genes with trait-associated expression were obtained by hybridising custom made application-specific cDNA microarrays with targets of discordant sib pairs of a porcine experimental population. Out of 238 genes addressed, nine genes were regulated by the factor>or=2 between the sib pairs. Differential gene expression was independently confirmed for selected genes by real time RT-PCR. Transcript levels of four genes (APOH, PEDF, SLCO1B3, TBG) were significantly different between the phenotype groups. Screening for trait associated markers within TBG and APOH by comparative sequencing of discordant sib pairs revealed a SNP at position nt 778 (A>C) (N229H) of TBG. No polymorphism in APOH was detected. Association analysis confirmed effects of TBG on carcass traits statistically. Allocating TBG to a QTL region on chromosome X revealed genetic evidence for the effect. Moreover, our results indicate that there are probably two polymorphisms segregating-one (N229H) altering binding capability of TBG and another still to be detected altering the transcription rate of TBG.

Analysis of Variance↗

PubMed2004

Polymorphisms of the porcine androgen receptor gene affecting its amino acid sequence and expression level.

Diverse physiological effects of the androgen receptor (AR), a nuclear transcription factor, and its mapping position within a quantitative trait loci (QTL) region on chromosome X propose it as an interesting candidate gene for pig reproduction and performance. Therefore, the aims of this study were isolation of the gene and detection of polymorphisms as a tool for association study and analysis of functional properties of the porcine AR. The mRNA and promoter sequences were obtained and screened for polymorphisms. Based on comparative sequencing, eight single nucleotide polymorphisms (SNPs), TG- and T-insertion/deletetion polymorphisms (INDELs) upstream transcription initiation sites, three SNPs in the 5'-untranslated region (UTR), one microsatellite (CCTTT)n in the intron of 5'-UTR, and a CAG-INDEL in exon 1 were detected. Two haplotypes originated from Duroc and Berlin Miniature Pig were segregating in the DUMI-F2 resource population. Characterization of the porcine AR promoter showed two conserved transcription start sites, a consensus sequence of GC-box and a homopurine/homopyrimidine stretch at similar locations compared to the human, rat and mouse as well as sequences similar to androgen response elements (ARE). The AR mRNA expression levels determined by real-time RT-PCR in various tissues of female pigs were high in ovary (100%) and adrenal gland (83.9% relative to ovary), moderate in uterus (61.6%) and liver (47.4%), and low in pituitary gland (1.3%) as well as in tonsil, muscle, mammary gland, leukocyte and jejunum (less than 1%). Detection of the AR mRNA transcripts in liver revealed that hemizygous males carrying the AR haplotype descended from Berlin Miniature pig had higher relative AR expressions than did those with the Duroc haplotype. Here we showed that the porcine AR is a highly polymorphic gene. Polymorphisms identified in the present study affect the predicted amino acid sequence as well as consensus transcription factor binding sites and are associated with the allele-specific differences of the AR mRNA transcript level in liver, reinforcing AR as a potential candidate gene for traits related to pig reproduction and performance.

5' Untranslated Regions↗

PubMed2004

Molecular cloning and sequencing of porcine C5 gene and its association with immunological traits.

The complement system helps in the lysis of invading pathogens and modulates the inflammatory as well as the humoral and cellular immune responses. C5 mediates many potent inflammatory and cytolytic events after proteolytic activation by complement convertase enzymes. Hence, to investigate the role of pig C5 ( pC5) as a candidate gene for disease resistance in pigs, the complete cDNA of pC5 was sequenced, screened for single nucleotide polymorphisms (SNPs), and an association analysis with various immunological parameters measured in F2 animals of a pig resource population based on a cross of Duroc and Berlin miniature pigs (DUMI) was carried out. In total, 5,422 bp of pC5 cDNA was sequenced, which codes for the 1,677-amino-acid precursor of C5. Four polymorphic sites were detected, one of which was segregating in the DUMI population in three genotypic patterns: AA, AC and CC. Classical (CH50) and alternative (AH50) complement activities, C3c levels, haptoglobin (HP) acute phase protein levels, and antibody titers against Mycoplasma (Mk) and Aujesky (ADV) vaccines were measured in the resource population. Association analysis between C5 and the immunological parameters was carried out using repeated measures mixed and general linear model analysis. The homozygote AA was found to be significantly different from the other two genotypes with respect to AH50 and CH50, whereas genotype CC was found to be significantly different from the other genotypes for C3c and HP levels. No significant difference could be seen between genotypes for antibody titers against vaccinations. Association of C5 with complement activity traits and acute phase proteins promotes pC5 as a candidate gene for innate disease resistance.

Acute-Phase Proteins↗

PubMed2004

BF, HP, DQB and DRB are associated with haemolytic complement activity, acute phase protein reaction and antibody response in the pig.

In order to examine the loci factor B (BF), C3, corticotropin releasing hormone (CRH), DQB, DRB, haptoglobin (HP) and transforming growth factor beta 1 (TGFB1) for association with traits of humoral, specific and unspecific defence F2-animals of a porcine resource family were genotyped at single nucleotide polymorphisms within these loci. Haemolytic complement activity in the alternative and classical pathway, C3c and haptoglobin serum concentration and antibody titres were determined immediately prior and at days 4 and 10 after vaccinations against Mycoplasma hyopneumoniae (Mh), Aujeszky's disease virus, and porcine reproductive and respiratory syndrome virus at 6, 14 and 16 weeks of age, respectively. Analysis of variance revealed association of BF, HP and DRB with C3c serum concentration. The trend of haemolytic complement activity and C3c serum concentration during the experiment was affected by the interaction of DQB genotype and time of measurement. Association with antibody titres were found for BF, DQB and DRB. Results of the mixed model analyses were confirmed by quantitative transmission disequilibrium test that showed linkage and association with antibody titres, complement activity and acute phase reaction at certain times of measurement. The findings promote the importance of the candidate genes for humoral mechanisms of unspecific and specific defence that provide natural resistance against many pathogens.

Acute-Phase Proteins↗

PubMed2004

Quantitative expression analysis of blastocyst-derived gene transcripts in preimplantation developmental stages of in vitro-produced bovine embryos using real-time polymerase chain reaction technology.

The main objective of the present study was to analyse the quantitative expression pattern of genes from a subtracted blastocyst transcriptome throughout the preimplantation developmental stages of in vitro-produced bovine oocytes and embryos. For this purpose, Day 5 morula (M) cDNAs were subtracted from Day 7 blastocyst (B) cDNAs (B-M) and used to establish a B-M subtracted cDNA library, as reported previously. From the total generated clones, 19 were analysed quantitatively. The mRNA samples isolated from pools of immature oocytes (n = 150), mature oocytes (n = 150) and two-cell (n = 80), four-cell (n = 40), eight-cell (n = 20), morula (n = 6) and blastocyst (n = 3) embryos were reverse transcribed and subjected to real-time polymerase chain reaction (PCR) using sequence-specific primers and SYBR green as the DNA dye. A relative standard curve method was used to analyse the real-time data taking the morula stage as a calibrator. Applying suppression subtractive hybridisation (SSH), a total of 71 clones, which represent 33 different expressed sequence tags, were generated and available for analysis. Most transcripts were analysed for the first time in bovine embryogenesis. The real-time PCR has validated the results of SSH positively for 84% (16/19) of transcripts, whereas 16% (3/19) showed deviation in the expression pattern from the one seen during SSH. Several transcript-specific expression patterns were observed for genes that play decisive roles in bovine embryogenesis. In addition to identification, accurately quantifying the expression profiles of transcripts during development will pave the way towards understanding the molecular mechanisms of embryogenesis and their potential role in early embryo development. Most importantly, the present study has contributed to the enrichment of bovine embryo gene collection by generating new transcripts involved in bovine embryo development.

Animals↗

PubMed2003

Molecular characterization of the pig C3 gene and its association with complement activity.

The complement system catalyzes direct lysis of micro-organisms and modulates phagocytosis, inflammation, humoral and cellular immune responses. Since the complement protein C3 is the central component within all pathways of complement activation, C3 is a candidate gene for complement activity and also for improved protection against many pathogens. The pig C3 gene was sequenced, screened for polymorphisms, and analyzed for association with hemolytic complement activity of the alternative and classical pathway (AH(50), CH(50)). C3c serum levels and haptoglobin (HP) serum concentrations were measured before and after vaccination against Mycoplasma hyopneumoniae, Aujeszky virus, and porcine reproductive and respiratory syndrome virus in F2 animals of a pig resource population based on crossbreeding of Duroc and Berlin Miniature Pig. The genomic C3 sequence covers 444 bp of promoter region, 41 exons and 40 introns, as well as 881 bp of the 3'-flanking region. The cDNA codes for a 1,661-amino acid precursor C3. Five polymorphic sites were detected in the 5'-UTR, intron 13, exon 15, exon 30, and the 3'-UTR. Within the resource population two haplotypes were found to segregate. Analysis of variance applying a repeated measures model revealed a significant effect of the interaction of C3 genotype and time of measurement relative to immunization on CH(50), AH(50,)and C3c that is likely to be due to variation of C3 expression. In contrast, the time course of the HP acute-phase reaction is not associated with C3 genomic variation. The association of C3 with complement activity indicates the importance of C3 as a candidate gene for natural resistance to micro-organisms, although the causative polymorphism modulating the expression of C3 remains to be delineated.

Alleles↗

PubMed2003

The GENETPIG database: a tool for comparative mapping in pig (Sus scrofa).

The GENETPIG database has been established for storing and disseminating the results of the European project: 'GENETPIG: identification of genes controlling economic traits in pig'. The partners of this project have mapped about 630 porcine and human ESTs onto the pig genome. The database collects the mapping results and links them to other sources of mapping data; this includes pig maps as well as available comparative mapping information. Functional annotation of the mapped ESTs is also given when a significant similarity to cognate genes was established. The database is accessible for consultation via the Internet at http://www.infobiogen.fr/services/Genetpig/.

Animals↗

PubMed2003

Identification and quantification of differentially expressed transcripts in in vitro-produced bovine preimplantation stage embryos.

In this study, we used mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) to analyze the mRNA expression patterns in in vitro-produced bovine 8-cell, 16-cell, morula, and blastocyst stage embryos and isolate differentially expressed amplicons. Moreover, we have used a fluorescence monitored real time quantitative PCR to quantify and analyze the expression patterns of the target differentially expressed transcripts through out the preimplantation stages from oocytes to blastocyst. For this, total RNA isolated from bovine 8-cell (n = 188), 16-cell (n = 94), morula (n = 35), and blastocyst (n = 15) were reverse transcribed and subjected to DDRT-PCR. Target differentially expressed transcripts were quantified by real time quantitative PCR. The cDNA banding pattern analysis revealed that large number of cDNA bands were conserved at 8-cell and blastocyst stage with a slight decrease at the morula stage. A total of 16 amplicons were cloned and sequenced. All expressed sequence tags (ESTs), except 1C19, showed sequence similarity with known genes or ESTs in GenBank. Sixty-two percent (10/16) of cDNA bands representing differentially expressed genes originated from 8-cell stage and the rest derived from the 16-cell, morula, or blastocyst stage. The quantitative PCR analysis has validated the expression patterns of 75% (12/16) of our transcripts to be in agreement with the results of DDRT-PCR. However, the quantitative PCR results of four transcripts showed a deviation from the pattern seen in DDRT-PCR. In conclusion, we have successfully applied mRNA DDRT-PCR to identify and isolate stage-specific expressed genes in bovine preimplantation embryos. In addition to validating the results of DDRT-PCR, quantitative real time PCR provides quantitative data on the expression of target genes.

Animals↗

PubMed2003

Association of the porcine C3 gene with haemolytic complement activity in the pig.

The complement component C3 plays an essential role in the activated complement system, which is involved in phagocytosis, inflammation and immunoregulation to destroy infectious microorganisms. The C3 molecule has more implications in the general defence mechanisms. In this study, the porcine C3 cDNA sequences including 5'- and 3'- flanking regions were determined and the polymorphisms in this gene were identified to carry out an association analysis between C3 and complement activity traits. Porcine C3 gene has high homology with human C3. Five single nucleotide polymorphisms (SNPs) and one microsatellite were detected in the porcine C3 gene. Haemolytic complement activity of alternative and classical pathways (ACH, CCP) was measured in 416 F2 animals of a crossbred of Duroc x Berlin Miniature Pig, which were immunized with Mycoplasma, Aujeszky and PRRS vaccines. C3 markers were found to be significantly associated (P <0.05) with both ACP and CCP. Animals with the more frequent haplotype present in Duroc and other commercial breeds exhibit higher ACP and CCP levels than the animals with haplotype specific to some Berlin Miniature Pigs. The association of C3 with complement activity reinforces the importance of C3 as a candidate gene for natural resistance to microorganisms.

Animals↗

PubMed2002

A source for expression profiling in single preimplantation bovine embryos.

Our knowledge of the genes active during normal preimplantation development in cattle is limited, despite the importance for further improvement of fertility and applicability of biotechniques, like in vitro production and embryo transfer. We report on the construction of cDNA libraries as a source for expression profiling in oocytes and single preimplantation cattle embryos. cDNAs were prepared from two unfertilized oocytes, single two-cell, four-cell and eight-cell, morula, and blastocyst stage embryos, respectively. The oocytes, eight-cell, morula, and blastocyst stage embryo-derived cDNAs were ligated to a lambda-based expression vector and these have complexities of 8 x 10(5), 5 x 10(5), 1 x 10(6) and 2 x 10(6) independent clones, respectively. A total of 48 clones were picked and sequenced, 62.5% (30/48) of the sequence were homologous to known transcripts from human and mouse, 18.75% (9/48) to expressed sequence tags (ESTs) of human and mouse origin. Novel sequences were detected at a frequency of 14.58% (7/48). PCR analyses of the embryonic libraries for specific genes revealed transcripts for genes including housekeeping genes (GAPDH and beta-actin), developmental genes (OCT-4, IGF-I receptor and homeodomain sequences) and genes coding for metabolic and protective enzymes (manganese superoxide dismutase, glutamine synthetase, flavin-containing mono-oxygenase, glutamate dehydrogenase, alpha-2-macroglobulin). These cDNA libraries are a valuable resource for the isolation of clones representing genes active at these early developmental stages. The ability to construct cDNA expression libraries from only a few cells will allow gene expression analyses from embryo biopsies and embryos derived by nuclear transfer procedures.

Animals↗

PubMed2002

Stage-specific expressed sequence tags obtained during preimplantation bovine development by differential display RT-PCR and suppression subtractive hybridization.

Differential display RT-PCR (DDRT-PCR) and suppression subtractive hybridization (SSH) were applied in order to detect preimplantation stage-specific expressed sequence tags (ESTs) of bovine embryos. Seventeen ESTs were detected from the differential display RT-PCR approach. All clones but two showed homology to genes or ESTs known in human, cattle or other species. One of the clones similar to H. sapiens mRNA for KIAA1764 protein was used exemplarily to quantify the transcripts by real-time PCR. The result of quantitative differential screening was found to be in agreement with DDRT-PCR banding patterns. In the second approach, a blastocyst-stage enriched cDNA library was constructed using SSH of blastocyst versus morula transcripts. The 71 clones that were analysed represent 33 distinct loci including candidate genes for the regulative processes during differentiation of inner cell mass (ICM) and trophoblast cells and the initial phase of embryo implantation, such as galectin-3 and fibronectin. As revealed by real-time PCR, the mRNA level of galectin-3 was three times higher in the blastocyst stage than in the morula stage. DDRT-PCR and SSH are both powerful tools for the identification of stage-specific expressed gene in preimplantation bovine embryos. Real-time PCR allows to test and confirm the outcome and to add quantitative data of selected transcripts of interest.

Animals↗

PubMed2002

Detection of quantitative trait loci for carcass traits in the pig by using AFLP.

For evaluation of the suitability of Amplified Fragment Length Polymorphism (AFLP) for detection of quantitative trait loci in farm animals, a combination of AFLP and selective genotyping has been applied as a rapid screening method for marker-QTL associations. Focusing on loci affecting eye muscle area, six extreme discordant sib pairs were selected from a Duroc x Berlin Miniature Pig F2 experimental cross and examined by using 48 AFLP primer combinations. Two prominent AFLP markers were converted into simple codominant PCR markers ( STS-Bo1 and STS-Bo3) and assigned to Sscr4 by physical and linkage mapping. Single marker analysis indicated association of the STS markers with a putative QTL influencing eye muscle area. Interval mapping confirmed the presence of a significant QTL for eye muscle area (Pgenomewide < 0.01) on the Sscr4, with STS-Bo1 being the closer marker. At the same location, significant effects (Pgenomewide < 0.01) on carcass length and backfat thickness were also detected. Our results demonstrate the capability of the combination of AFLP analysis and selective genotyping as a method for detection of genome regions containing QTL in livestock.

Animals↗