Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

Transcriptome Profiling of <em>In-Vivo</em> Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

University of Alberta

In this paper we analyzed 49 lactate dehydrogenase (LDH) sequences, mostly from vertebrates. The amino acid sequence differences were found to be larger for a human-killifish pair than a human-lamprey pair. This indicates that some protein sequence convergence may occur and reduce the sequence differences in distantly related species. We also examined transitions and transversions separately for several species pairs and found that the transitions tend to be saturated in the distantly related species pair, while transversions are increasing. We conclude that transversions maintain a conservative rate through the evolutionary time. Kimura's two-parameter model for multiple-hit correction on transversions only was used to derive a distance measure and then construct a neighbor-joining (NJ) tree. Three findings were revealed from the NJ tree: (i) the branching order of the tree is consistent with the common branch pattern of major vertebrates; (ii) Ldh-A and Ldh-B genes were duplicated near the origin of vertebrates; and (iii) Ldh-C and Ldh-A in mammals were produced by an independent gene duplication in early mammalian history. Furthermore, a relative rate test showed that mammalian Ldh-C evolved more rapidly than mammalian Ldh-A. Under a two-rate model, this duplication event was calibrated to be approximately 247 million years ago (mya), dating back to the Triassic period. Other gene duplication events were also discovered in Xenopus, the first duplication occurring approximately 60-70 mya in both Ldh-A and Ldh-B, followed by another recent gene duplication event, approximately 20 mya, in Ldh-B.

Phylogenetic analysis of vertebrate lactate dehydrogenase (LDH) multigene families.

Vascular endothelial growth factor (VEGF) is a key regulator for placental angiogenesis and vascular functions via activating two high affinity tyrosine-kinase receptors, VEGF receptor-1 (VEGFR-1) and -2 (VEGFR-2). Recently, a specific VEGF165 receptor, neuropilin-1 (NP-1), was also identified in endothelial cells and upon VEGF binding, NP-1, synergistically with VEGFR-2, enhances VEGF-induced cell proliferation and migration. To evaluate the role of VEGF and NP-1 in regulating fetoplacental angiogenesis and endothelial function, an ovine fetal placental artery endothelial (OFPAE) cell line was established. In this study, an OFPAE cell cDNA library was constructed. Two positive clones for VEGF and one for NP-1 were isolated from the OFPAE cell cDNA library, and their partial 3' sequences were identified. The sequence of VEGF cDNA insert had 98% homology to the reported ovine VEGF (GenBank accesssion # X89506). The partial NP-1 cDNA sequence included a portion of the protein coding region and a complete 3' untranslated region (UTR), and had 90% homology to human NP-1 (GenBank accession # AF016050). The predicted amino acid sequence of ovine NP-1 was 97-98% identical to human (GenBank accession # AAC12921.1), mouse (GenBank accession # NP_032763), and rat (GenBank accession # AAC53345.1) NP-1. Two CU-rich stabilizing and two consensus destabilizing elements 5'-AUUUA-3' were identified in the 3' UTR of ovine NP-1 cDNA sequence. These elements are the potential binding sites for mRNA-binding proteins which may regulate the stability of NP-1 mRNA. Expression of VEGF and NP-1 in OFPAE cells and fetal placentas was confirmed by Northern and Western blot analyses. Using PCR analysis, we also identified partial sequences of multiple VEGF isoforms (VEGF188, 183, 164, and 120) as well as VEGFR-1, VEGFR-2, and neuropilin-2 (NP-2) from the OFPAE cell cDNA library. These results indicate that multiple isoforms of VEGF are expressed in OFPAE cells. Moreover, we also identified, for the first time, a complete 3' UTR of NP-1 cDNA in any species. Together with expression of VEGF and VEGF receptors in OFPAE cells, we propose that there is an autocrine mechanism by which VEGF regulates fetal placental angiogenesis and other functions of endothelial cells.

Co-expression of vascular endothelial growth factor and neuropilin-1 in ovine feto-placental artery endothelial cells.

Antimicrobial peptides play a crucial role as the first line of defense against invading pathogens. Several types of antimicrobial peptides have been isolated from fish, mostly of the cationic alpha-helical variety. Here, we present the cDNA sequences of five highly disulphide-bonded hepcidin-like peptides from winter flounder, Pseudopleuronectes americanus (Walbaum) and two from Atlantic salmon, Salmo salar (L.). These hepcidin-like molecules consist of a 24 amino acid signal peptide and an acidic propiece of 38-40 amino acids in addition to the mature processed peptide of 19-27 amino acids. Exhaustive data mining of GenBank with these sequences revealed that similar peptides are encoded in the genomes of Japanese flounder, rainbow trout, hybrid striped bass and medaka, indicating that they are widespread among fish. Southern hybridization analysis suggests that closely related hepcidin-like genes are present in other flatfish species, and that they exist as a multigene family clustered on the winter flounder genome. Hepcidin variants are differentially expressed during bacterial challenge, during larval development of P. americanus and in different tissues of adult fish.

Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish.

Commercially available human complementary DNA microarrays were used to compare differential expression in the livers of Atlantic salmon ( Salmo salar) infected with Aeromonas salmonicida and of healthy fish. Complementary DNA probes were prepared from total RNA isolated from livers of control salmon and infected salmon by reverse transcription in the presence of (33)P-dCTP and independently hybridized to human GENE-FILTERS GF211 microarrays. Of the 4131 known genes on the microarray, 241 spots gave clearly detectable signals using labeled RNA from the control salmon liver. Of these, 4 spots were consistently found to have a greater than 2-fold increase in infected salmon compared with controls when using the same pair of filters to generate hybridization data from triplicates. These up-regulated genes were ADP/ATP translocase (AAT2), Na(+)/K(+) ATPase, acyloxyacyl hydrolase (AOAH), and platelet-derived growth factor (PDFG-A). A BlastN search revealed an AAT2 homolog from Atlantic salmon, and a reverse transcriptase polymerase chain reaction assay using primers based on this sequence confirmed its up-regulation (approx. 1.8-fold) during early infection. This work demonstrates the feasibility of using human microarrays to facilitate the discovery of differentially expressed genes in Atlantic salmon, for which no homologous microarrays are available.

Use of human cDNA microarrays for identification of differentially expressed genes in Atlantic salmon liver during Aeromonas salmonicida infection.

Third-trimester human placentas from normal and preeclamptic pregnancies were evaluated for possible changes in gene expression patterns by microarray analysis.

cDNA microarray analysis of gene expression profiles in human placenta: up-regulation of the transcript encoding muscle subunit of glycogen phosphorylase in preeclampsia.

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida.

The response of Atlantic salmon, Salmo salar, to infection by the bacterial pathogen Aeromonas salmonicida (the causative agent of furunculosis), was investigated using a cohabitation model and a custom Atlantic salmon cDNA microarray consisting of over 4000 different amplicons. Pooled samples of each of three immune-relevant tissues (spleen, head kidney and liver) were obtained from fish exposed to infected salmon for 13 days. Reverse transcription-PCR assays were used to verify the differential expression of 12 candidate genes uncovered by microarray analysis. Among the differentially expressed genes were several previously revealed by suppression subtractive hybridization and EST surveys and that are recognized to encode humoral components of the innate immune system. Other genes identified in this study were not previously associated with infection. In addition, a number of genes with no known homologs were uncovered. Determination of their specific roles during infection may lead to a better understanding of innate immunity.

Identification of genes differentially expressed in Atlantic salmon (Salmo salar) in response to infection by Aeromonas salmonicida using cDNA microarray technology.

Diapause-destined embryos of the crustacean Artemia franciscana cease development as gastrulae, encyst, and enter a resting stage characterized by greatly reduced metabolic activity and extreme stress resistance. To better understand diapause induction and maintenance in Artemia embryos gene expression was analyzed by subtractive hybridization at two days post-fertilization, a time early in this developmental process. Eighty-five of 264 cDNA clones sequenced matched GenBank entries and they fell into categories designated as environmental information processing, cellular processes, genetic information processing and metabolism. Semi-quantitative RT-PCR of cDNAs populating the subtractive library identified seventeen up-regulated and four down-regulated transcripts, the former including those encoding a human transcription cofactor homologue, three small heat shock proteins, putative cell growth suppressor proteins and several enzymes. As examples, p8 may modulate gene expression during diapause in Artemia embryos. BRCA1 associated protein-1 (BAP1) and other functionally related proteins may influence cell growth and division during transition into diapause, a time when these processes are inhibited, whereas small heat shock proteins protect embryos from stress. This study represents the first systematic molecular characterization of diapause in crustaceans. Several differentially expressed genes were identified, expanding the repertoire of proteins potentially modified during diapause and suggesting mechanistic pathways indigenous to the initiation and maintenance of this physiological state.

Gene expression in diapause-destined embryos of the crustacean, Artemia franciscana.

Transcriptomic research using microarrays and RNA-Sequencing (RNA-seq) is now possible starting from minute biological samples, such as clinical specimens or embryos, due to the development of highly sensitive and reproducible cDNA synthesis methods. Here, we describe a quick method of RNA amplification and double-stranded cDNA synthesis starting with 10 ng of high-quality total RNA extracted from porcine embryos. The final product (double-stranded DNA) is adequate for the detection by RNA-seq of protein-coding transcripts, as well as of all the other classes of noncoding RNAs, including pseudogenes.

Stephen C.M. Tsoi

Department of Agricultural,
Food and Nutritional Science,
Department of Agricultural, Food and Nutritional Science

University of Alberta

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Stephen C.M. Tsoi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Agricultural,Food and Nutritional Science,Department of Agricultural, Food and Nutritional Science

University of Alberta

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Department of Agricultural,
Food and Nutritional Science,
Department of Agricultural, Food and Nutritional Science