Quantification of Fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos

<p>Abstract</p> <p>Background</p> <p>Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development.</p> <p>Results</p> <p>RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells.</p> <p>In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 α integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 β integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV).</p> <p>Conclusion</p> <p>The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.</p

Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A

BACKGROUND: An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor γ coactivator 1α (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. RESULTS: The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B), the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05). CONCLUSION: This study provides a new set of reference genes (ACTB, TBP and TOP2B) suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass

Characterization of the genomic region containing the Shadow of Prion Protein (SPRN) gene in sheep

<p>Abstract</p> <p>Background</p> <p>TSEs are a group of fatal neurodegenerative diseases occurring in man and animals. They are caused by prions, alternatively folded forms of the endogenous prion protein, encoded by <it>PRNP</it>. Since differences in the sequence of <it>PRNP </it>can not explain all variation in TSE susceptibility, there is growing interest in other genes that might have an influence on this susceptibility. One of these genes is <it>SPRN</it>, a gene coding for a protein showing remarkable similarities with the prion protein. Until now, <it>SPRN </it>has not been described in sheep, a highly relevant species in prion matters.</p> <p>Results</p> <p>In order to characterize the genomic region containing <it>SPRN </it>in sheep, a BAC mini-contig was built, covering approximately 200,000 bp and containing the genes <it>ECHS1</it>, <it>PAOX</it>, <it>MTG1</it>, <it>SPRN</it>, <it>LOC619207, CYP2E1 </it>and at least partially <it>SYCE1</it>. FISH mapping of the two most exterior BAC clones of the contig positioned this contig on Oari22q24. A fragment of 4,544 bp was also sequenced, covering the entire <it>SPRN </it>gene and 1206 bp of the promoter region. In addition, the transcription profile of <it>SPRN </it>in 21 tissues was determined by RT-PCR, showing high levels in cerebrum and cerebellum, and low levels in testis, lymph node, jejunum, ileum, colon and rectum.</p> <p>Conclusion</p> <p>Annotation of a mini-contig including <it>SPRN </it>suggests conserved linkage between Oari22q24 and Hsap10q26. The ovine <it>SPRN </it>sequence, described for the first time, shows a high level of homology with the bovine, and to a lesser extent with the human <it>SPRN </it>sequence. In addition, transcription profiling in sheep reveals main expression of <it>SPRN </it>in brain tissue, as in rat, cow, man and mouse.</p

Comparative analysis of a BAC contig of porcine chromosome 13q31-q32 and human chromosome 3q21-q22

BACKGROUND: The gene(s) encoding the ETEC F4ab/ac receptors, involved in neonatal diarrhoea in pigs (a disease not yet described in humans), is located close to the TF locus on Sscr13. In order to reveal and characterize possible candidate genes encoding these receptors, a porcine physical map of the TF region is indispensable. RESULTS: A contig of 33 BAC clones, covering approximately 1.35 Mb surrounding the TF locus on Sscr13q31-q32, was built by chromosome walking. A total of 22,552 bp from the BAC contig were sequenced and compared with database sequences to identify genes, ESTs and repeat sequences, and to anchor the contig to the syntenic region of the human genome sequence (Hsap3q21-q22). The contig was further annotated based on this human/porcine comparative map, and was also anchored to the Sanger porcine framework map and the integrated map of Sscr13 by RH mapping. CONCLUSION: The annotated contig, containing 10 genes and 2 ESTs, showed a complete conservation of linkage (gene order and orientation) with the human genome sequence, based on 46 anchor points. This underlines the importance of the human/porcine comparative map for the identification of porcine genes associated with genetic defects and economically important traits, and for assembly of the porcine genome sequence

Identification and expression analysis of genes associated with bovine blastocyst formation

<p>Abstract</p> <p>Background</p> <p>Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation.</p> <p>First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of <it>in vitro </it>embryo production procedures.</p> <p>Results</p> <p>A subtractive cDNA library was constructed enriched for transcripts preferentially expressed at the blastocyst stage compared to the 2-cell and 8-cell stage. Sequence information was obtained for 65 randomly selected clones. The RNA expression levels of 12 candidate genes were determined throughout 3 stages of preimplantation embryo development (2-cell, 8-cell and blastocyst) and compared with the RNA expression levels of <it>in vivo </it>"golden standard" embryos using real-time PCR. The RNA expression profiles of 9 (75%) transcripts (<it>KRT18</it>, <it>FN1</it>, <it>MYL6</it>, <it>ATP1B3</it>, <it>FTH1</it>, <it>HINT1</it>, <it>SLC25A5</it>, <it>ATP6V0B</it>, <it>RPL10</it>) were in agreement with the subtractive cDNA cloning approach, whereas for the remaining 3 (25%) (<it>ACTN1</it>, <it>COPE</it>, <it>EEF1A1</it>) the RNA expression level was equal or even higher at the earlier developmental stages compared to the blastocyst stage. Moreover, significant differences in RNA expression levels were observed between <it>in vitro </it>and <it>in vivo </it>produced embryos. By immunofluorescent labelling, the protein expression of KRT18, FN1 and MYL6 was determined throughout bovine preimplantation embryo development and showed the same pattern as the RNA expression analyses.</p> <p>Conclusion</p> <p>By subtractive cDNA cloning, candidate genes involved in blastocyst formation were identified. For several candidate genes, important differences in gene expression were observed between <it>in vivo </it>and <it>in vitro </it>produced embryos, reflecting the influence of the <it>in vitro </it>culture system on the embryonic gene expression. Both RNA and protein expression analysis demonstrated that <it>KRT18</it>, <it>FN1 </it>and <it>MYL6 </it>are differentially expressed during preimplantation embryo development and those genes can be considered as markers for bovine blastocyst formation.</p

Characterization of the ovine ribosomal protein SA gene and its pseudogenes

Background: The ribosomal protein SA (RPSA), previously named 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR) is a multifunctional protein that plays a role in a number of pathological processes, such as cancer and prion diseases. In all investigated species, RPSA is a member of a multicopy gene family consisting of one full length functional gene and several pseudogenes. Therefore, for studies on RPSA related pathways/pathologies, it is important to characterize the whole family and to address the possible function of the other RPSA family members. The present work aims at deciphering the RPSA family in sheep. Results: In addition to the full length functional ovine RPSA gene, 11 other members of this multicopy gene family, all processed pseudogenes, were identified. Comparison between the RPSA transcript and these pseudogenes shows a large variety in sequence identities ranging from 99% to 74%. Only one of the 11 pseudogenes, i.e. RPSAP7, shares the same open reading frame (ORF) of 295 amino acids with the RPSA gene, differing in only one amino acid. All members of the RPSA family were annotated by comparative mapping and fluorescence in situ hybridization (FISH) localization. Transcription was investigated in the cerebrum, cerebellum, spleen, muscle, lymph node, duodenum and blood, and transcripts were detected for 6 of the 11 pseudogenes in some of these tissues. Conclusions: In the present work we have characterized the ovine RPSA family. Our results have revealed the existence of 11 ovine RPSA pseudogenes and provide new data on their structure and sequence. Such information will facilitate molecular studies of the functional RPSA gene taking into account the existence of these pseudogenes in the design of experiments. It remains to be investigated if the transcribed members are functional as regulatory non-coding RNA or as functional proteins

Reference Gene Selection for Insect Expression Studies Using Quantitative Real-Time PCR: The Head of the Honeybee, Apis mellifera, After a Bacterial Challenge

In this study an important and often neglected aspect of gene expression studies in insects, the validation of appropriate reference genes with stable expression levels between sample groups, is addressed. Although in this paper the reference gene selection for the honeybee, Apis mellifera L. (Hymenoptera: Apidae) head was tested in the context of bacterial challenge with Escherichia coli, this work can serve as a resource to help select and screen insect reference genes for gene expression studies in any tissue and under any experimental manipulation. Since it is recommended to use multiple reference genes for accurate normalization, we analyzed the expression of eleven candidate reference genes in the honeybee head, for their potential use in the analysis of differential gene expression following bacterial challenge. Three software programs, BestKeeper, Normfinder and geNorm, were used to assess candidate reference genes. GeNorm recommended the use of four reference genes. Both geNorm and Normfinder identified the genes GAPDH, RPS18, actin and RPL13a as the most stable ones, only differing in their ranking order. BestKeeper identified RPS18 as being the reference gene with the least overall variation, but also actin and GAPDH were found to be the second and third most stable expressed gene. By a combination of three software programs the genes actin, RPS18 and GAPDH were found suitable reference genes in the honeybee head in the context of bacterial infection

A dual fluorescent multiprobe assay for prion protein genotyping in sheep

BACKGROUND: Scrapie and BSE belong to a group of fatal, transmissible, neurodegenerative diseases called TSE. In order to minimize the risk of natural scrapie and presumed natural BSE in sheep, breeding programmes towards TSE resistance are conducted in many countries based on resistance rendering PRNP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (R/H/Q). Therefore, a reliable, fast and cost-effective method for routine PRNP genotyping in sheep, applicable in standard equipped molecular genetic laboratories, will be a vital instrument to fulfill the need of genotyping hundreds or thousands of sheep. METHODS: A dual fluorescent multiprobe assay consisting of 2 closed tube PCR reactions containing respectively 4 and 3 dual-labelled fluorescent ASO probes for the detection in real-time of the 7 allelic variants of sheep PRNP mentioned above. RESULTS: The assay is succesfully performed using unpurified DNA as a template for PCR, without any post-PCR manipulations and with semi-automatic determination of the PRNP genotypes. The performance of the assay was confirmed via PCR-RFLP and sequencing in a cross-validation study with 50 sheep. CONCLUSIONS: We report the development and validation of a robust, reliable and reproducible method for PRNP genotyping of a few to many sheep samples in a fast, simple and cost-effective way, applicable in standard equipped molecular genetic laboratories. The described primer/probe design strategy can also be applied for the detection of other polymorphisms or disease causing mutations

Short-chain fructo-oligosaccharides supplementation to suckling piglets : assessment of pre- and post-weaning performance and gut health

Farmers face difficulties in redeeming their investment in larger litter sizes since this comes with larger litter heterogenicity, lower litter resilience and risk of higher mortality. Dietary oligosaccharides, given to the sow, proved beneficial for the offspring's performance. However, giving oligosaccharides to the suckling piglet is poorly explored. Therefore, this field trial studied the effect of dietary short-chain fructo-oligosaccharides (scFOS; 1g/day; drenched) supplementation to low (LBW, lower quartile), normal (NBW, two intermediate quartiles) and high (HBW, upper quartile) birth weight piglets from birth until 7 or 21 days of age. Performance parameters, gut microbiome and short-chain fatty acids profile of feces and digesta were assessed at birth (d 0), d 7, weaning (d 21.5) and 2 weeks post-weaning (d 36.5). Additional parameters reflecting gut health (intestinal integrity and morphology, mucosal immune system) were analysed at d 36.5. Most parameters changed with age or differed with the piglet's birth weight. Drenching with scFOS increased body weight by 1 kg in NBW suckling piglets and reduced the post-weaning mortality rate by a 100%. No clear difference in the IgG level, the microbiota composition and fermentative activity between the treatment groups was observed. Additionnally, intestinal integrity, determined by measuring intestinal permeability and regenerative capacity, was similar between the treatment groups. Also, intestinal architecture (villus lenght, crypt depth) was not affected by scFOS supplementation. The density of intra-epithelial lymphocytes and the expression profiles (real-time qPCR) for immune system-related genes (IL-10, IL-1ss, IL-6, TNF alpha and IFN gamma) were used to assess mucosal immunity. Only IFN gamma expression, was upregulated in piglets that received scFOS for 7 days. The improved body weight and the reduced post-weaning mortality seen in piglets supplemented with scFOS support the view that scFOS positively impact piglet's health and resilience. However, the modes of action for these effects are not yet fully elucidated and its potential to improve other performance parameters needs further investigation