Biomedical subjects

Christopher K Tuggle

Publications and source records attributed to Christopher K Tuggle.

11 recordsLinked to original sources

PubMed2006

Gene expression profiling in Salmonella Choleraesuis-infected porcine lung using a long oligonucleotide microarray.

Understanding the transcriptional response to pathogenic bacterial infection within food animals is of fundamental and applied interest. To determine the transcriptional response to Salmonella enterica serovar Choleraesuis (SC) infection, a 13,297-oligonucleotide swine array was used to analyze RNA from control, 24-h postinoculation (hpi), and 48-hpi porcine lung tissue from pigs infected with SC. In total, 57 genes showed differential expression (p < 0.001; false discovery rate = 12%). Quantitative real-time PCR (qRT-PCR) of 61 genes was used to confirm the microarray results and to identify pathways responding to infection. Of the 33 genes identified by microarray analysis as differentially expressed, 23 were confirmed by qRT-PCR results. A novel finding was that two transglutaminase family genes (TGM1 and TGM3) showed dramatic increases in expression postinoculation; combined with several other apoptotic genes, they indicated the induction of apoptotic pathways during SC infection. A predominant T helper 1-type immune response occurred during infection, with interferon gamma (IFNG) significantly increased at 48 hpi. Genes induced by IFNs (GBP1, GBP2, C1S, C1R, MHC2TA, PSMB8, TAP1, TAP2) showed increased expression during porcine lung infection. These data represent the first thorough investigation of gene regulation pathways that control an important porcine respiratory and foodborne bacterial infection.

Animals↗

PubMed2005

Analysis of porcine differential gene expression following challenge with Salmonella enterica serovar Choleraesuis using suppression subtractive hybridization.

Swine-adapted Salmonella enterica subsp. enterica serovar Choleraesuis (S. Choleraesuis) is the pathogen most frequently isolated from diseased pigs and may affect host gene expression in a species-specific manner. To characterize the porcine transcriptional response to S. Choleraesuis infection, the mRNA profiles from the mesenteric lymph nodes of three non-infected and three experimentally infected pigs at 24 h post-inoculation were analyzed by suppression subtractive hybridization (SSH). Forty-four up-regulated and 44 down-regulated genes were revealed by differential cDNA screening of 384 forward and 288 reverse subtracted cDNA clones. The DNA sequence of the cDNA clones identified genes with a role in a variety of cellular functions as well as gene products of unknown function. Seven up-regulated genes (CXCL10, CXCR4, SDCBP, DNAJA1, HSPH1, HSP90 and ANXA5) and two functionally related genes (HSP70 and DNAJA4:pDJA1) were selected for further analysis based on their predicted roles in infection and immunity. Real-time RT-PCR was performed using RNA collected from a time course of infection spanning from the acute phase (8 h) to the chronic phase (21 days) to confirm and quantitate the up-regulation of the SSH-enriched genes. Correlating with the clinical signs of infection (fever, diarrhea and lethargy), the most dramatic induction of gene expression for all nine genes occurred at 48 h post-inoculation. This investigation further defines the porcine response to a host-adapted strain of Salmonella by revealing the differential expression of genes with a role in a variety of host cellular functions including innate immunity and cytoskeleton regulation.

Animals↗

PubMed2005

Ectopic HOXA5 expression results in abnormal differentiation, migration and p53-independent cell death of superficial dorsal horn neurons.

Previously, we reported a line of mice (Hoxa5SV2) that ectopically expresses HOXA5 in the developing cervical and brachial dorsal spinal cord. Animals from this line exhibited a clear loss of cells in the outer lamina of the mature dorsal horn that coincided with an adult phenotype of sensory and motor defects of the forelimb. In this report, we examined the etiology of lost dorsal horn cells. Cells normally fated to populate the outer laminae I-III of the dorsal horn migrated inappropriately, as the percentage of laterally positioned cells in the dorsal horn was significantly reduced in Hoxa5SV2 transgenics. Apoptosis was a major cause of cell loss while proliferation of neurons was not affected in Hoxa5SV2 animals. Although Hoxa5 has been shown in vitro to regulate p53 expression and cause p53-dependent apoptosis, p53 was not required in vivo for the inappropriate apoptosis seen in Hoxa5SV2 mice, or for the normal death of motor neurons. Normal apoptosis is not dependent on Hoxa5, as the level of ventral horn motor neuron apoptosis was not changed in Hoxa5 null animals. As a possible cause of aberrant migration and/or apoptosis of dorsal neurons, misexpression of cell type markers was demonstrated. Further, the expression pattern of laminar markers was altered and sensory fibers aberrantly penetrated the outer lamina of mutants. Our evidence suggests that the loss of dorsal horn neurons in Hoxa5SV2 mutants was due to misexpression of dorsal horn neuronal markers, aberrant migration, and inappropriate apoptosis.

Animals↗

PubMed2005

Transcriptional profiling using a novel cDNA array identifies differential gene expression during porcine embryo elongation.

A novel porcine cDNA array, containing 1,015 PCR products selected for embryonic expression, was used for transcriptional profiling of conceptuses at four stages of peri-implantation development. Total conceptus RNA from small spherical, large spherical, tubular, and filamentous stages was amplified, converted to cDNA, and hybridized to membranes. Initially, normalized signal intensities obtained using cDNA from total RNA or from amplified RNA were compared. Uniform distribution of P-values associated with t-tests conducted for each gene indicated no evidence that amplification introduced bias. Analysis of data obtained by using amplified targets and the novel array identified genes differentially expressed across stages. Such genes were identified by testing for significant stage effects in gene-specific mixed models. A total of nine genes were declared differentially expressed. Six of the nine genes had P-values less than 0.001, and a false discovery rate of approximately 17% was associated with this significance threshold. Two out of six genes were significant when using the Bonferroni method to control the probability of one or more false positives. The other three genes had P-values between 0.001 and 0.01 and exhibited differences greater than twofold between stages. All four genes selected for confirmation (steroidogenic acute regulatory protein, interleukin 1 beta, transforming growth factor beta 3, and thymosin beta 10) were shown to be differentially expressed by using quantitative real time RT-PCR. Our study shows that RNA amplification is useful for transcriptional profiling with limiting porcine embryonic RNA, and that this novel targeted array can detect differential gene expression during trophoblastic elongation. Finally, our results contribute to an increased understanding of the temporal patterns of expression of known genes controlling conceptus development, as well as identify novel genes also differentially regulated during implantation.

Animals↗

PubMed2005

Dynamic expression of murine HOXA5 protein in the central nervous system.

The Hox genes encode transcription factors that are indispensable for proper spatio-temporal patterning of the vertebrate body axes. As for other Hox genes, region-specific expression of Hoxa5 appears to be important for correct function during development. In mouse, Hoxa5 transcripts are differentially expressed in specific mesoderm-derived structures and in the most anterior domain of expression in the central nervous system (CNS), in contrast to indistinct patterns seen in the posterior CNS. However, the functional significance of any pattern of protein-coding RNAs must be verified by correlating the presence of the protein(s) and RNAs. Here, we describe the dynamic pattern of HOXA5 protein during mouse embryogenesis. The HOXA5 protein is detected as early as embryonic day (E) 9.0, and is found, as development proceeds, in several mesoderm-derived structures such as prevertebrae (pv), proximal forelimb bud, scapula, lung, trachea, and gut. In addition, the protein shows a strikingly restricted and dynamic expression pattern in the developing CNS, and is detected in both motor neurons and interneurons between E10.5 and E13.5. Moreover, this CNS region-specific HOXA5 protein pattern is more restricted than the pattern observed for the Hoxa5 transcripts. In many mesoderm-derived tissues affected by the Hoxa5 mutation, the expression pattern of HOXA5 protein corresponds to that of the putative functional Hoxa5 transcript. However, in the CNS, this correlation is exclusively demonstrated in the most anterior domain of expression. Overall, the HOXA5 protein pattern is consistent with its proposed role in positional specification in mesodermal structures, as well as in the embryonic neuraxis.

Animals↗

PubMed2005

Validation of a first-generation long-oligonucleotide microarray for transcriptional profiling in the pig.

A first-generation porcine oligonucleotide set, representing 13,297 cDNAs and ESTs, has been designed by Qiagen-Operon for transcriptional profiling. To validate this set, microarrays containing each 70-mer oligonucleotide, referred to as the Qiagen-NRSP8 array, were hybridized with targets from porcine adult liver, lung, muscle, or small intestine. Transcriptome analyses showed that 11,328 of the oligonucleotides demonstrated expression in at least one tissue. Statistical analyses revealed that 1810 genes showed differential expression among tissues (Bonferroni adjusted p < 0.05). Biological pathways identified by DAVID/EASE analysis using a list of 423 tissue-selective genes matched archetypal pathways in the corresponding human or mouse tissue. Real-time quantitative PCR confirmed expression patterns for 9 of 11 genes tested. Our results demonstrate that this first-generation porcine oligonucleotide array is informative and the specificity is high. This is essential validation for investigators using the Qiagen-NRSP8 array for porcine functional genomics and for using the pig in modeling important physiological problems.

Animals↗

PubMed2005

Cdx protein interaction with Hoxa5 regulatory sequences contributes to Hoxa5 regional expression along the axial skeleton.

Hox gene functions are intimately linked to correct developmental expression of the genes. The identification of cis-acting regulatory sequences and their associated trans-acting factors constitutes a key step in deciphering the mechanisms underlying the correct positioning of the functional domain of Hox genes along the anterior-posterior axis. We have identified DNA elements driving Hoxa5 regionalized expression in mice, using the 2.1-kb mesodermal enhancer (MES) localized in Hoxa5 3' flanking sequences as a starting point. The MES sequence comprises regulatory elements targeting Hoxa5 expression in the limbs, the urogenital and gastrointestinal tracts, and the cervical-upper thoracic region of the prevertebral column. A 164-bp DNA fragment within the MES caudally restricts Hoxa5 expression at the level of prevertebra 10, corresponding to the posterior limit of its functional domain. Cdx proteins directly bind to this element in vitro via two conserved sites. Preventing Cdx binding by mutating the sites causes caudal expansion of the transgene expression domain. Of all three murine Cdx proteins that bind this element in vitro, Cdx4 has emerged as a potential regional posterior repressor of Hoxa5 expression. The restrictive control provided by Cdx interactions with Hoxa5 regulatory sequences may be one of the critical events in cervicothoracic axial specification.

Animals↗

PubMed2004

Transgenic mice ectopically expressing HOXA5 in the dorsal spinal cord show structural defects of the cervical spinal cord along with sensory and motor defects of the forelimb.

Mutation of murine Hoxa5 has shown that HOXA5 controls lung, gastrointestinal tract and vertebrae development. Hoxa5 is also expressed in the spinal cord, yet no central nervous system phenotype has been described in Hoxa5 knockouts. To identify the role of Hoxa5 in spinal cord development, we developed transgenic mice that express HOXA5 in the dorsal spinal cord in the brachial region. Using HOXA5-specific antibodies, we show this expression pattern is ectopic as the endogenous protein is expressed only in the ventral spinal cord at this anterio-posterior level. This transgenic line (Hoxa5SV2) also displays forelimb-specific motor and sensory defects. Hoxa5SV2 transgenic mice cannot support their body weight in a forelimb hang, and forelimb strength is decreased. However, Rotarod performance was not impaired in Hoxa5SV2 mice. Hoxa5SV2 mice also show a delayed forelimb response to noxious heat, although hindlimb response time was normal. Administration of an analgesic significantly reduced the hang test defect and decreased the transgene effect on forelimb strength, indicating that pain pathways may be affected. The morphology of transgenic cervical (but not lumbar) spinal cord is highly aberrant. Nissl staining indicates superficial laminae of the dorsal horn are severely disrupted. The distribution of cells and axons immunoreactive for substance P, neurokinin-B, and their primary receptors were aberrant only in transgenic cervical spinal cord. Further, we see increased levels of apoptosis in transgenic spinal cord at embryonic day 13.5. Our evidence suggests apoptosis due to HOXA5 misexpression is a major cause of loss of superficial lamina cells in Hoxa5SV2 mice.

Animals↗

PubMed2004

High-resolution comparative mapping of pig Chromosome 4, emphasizing the FAT1 region.

The first quantitative trait locus (QTL) in pigs, FAT1, was found on Chromosome 4 (SSC4) using a Wild Boar intercross. Further mapping has refined the FAT1 QTL to a region with conserved synteny to both human Chromosomes 1 and 8. To both improve the comparative map of the entire SSC4 and to define the specific human chromosome region with conserved synteny to FAT1, we have now mapped 103 loci to pig Chromosome 4 using a combination of radiation hybrid and linkage mapping. The physical data and linkage analysis results are in very good agreement. Comparative analysis revealed that gene order is very well conserved across SSC4 compared to both HSA1 and HSA8. The breakpoint in conserved synteny was refined to an area of about 23 cR on the q arm of SSC4 corresponding to a genetic distance of less than 0.5 cM. Localizations of the centromeres do not seem to have been conserved between the two species. No remnants of the HSA1 centromere were detected on the corresponding region on SSC4 and traces from the centromeric region of SSC4 cannot clearly be revealed on the homologous region on HSA8. This refined SSC4 map and the comparative analysis will be a great aid in the search for the genes underlying the FAT1 locus.

Animals↗

PubMed2004

PLET1 (C11orf34), a highly expressed and processed novel gene in pig and mouse placenta, is transcribed but poorly spliced in human.

Sequencing of porcine cDNAs identified a novel EST with high frequency in placenta tissue. Full-length PLET1 (placenta-expressed transcript 1, also called C11orf34) matched a mouse cDNA and many bovine and mouse ESTs but no human transcripts or ESTs. However, the porcine cDNA matched several putative exons within a human genomic DNA fragment on chromosome 11. This human locus is in a region of conserved synteny with pig chromosome 9, to which the porcine gene was subsequently mapped. RNA blot hybridization showed that this gene had high expression in porcine and mouse conceptus and throughout placenta development. In situ hybridization using mouse placenta showed PLET1 expression in trophoblast cells of the labyrinth, as well as in spongiotrophoblast and glycogen trophoblast cells. However, no expression of PLET1 was detected by RNA blot analysis of human placenta, although RT-PCR analysis detected very small amounts of partially spliced RNA that were significantly less abundant than the RNA levels in mouse placenta. Donor and acceptor splicing site sequences in the exons of the human gene are poorly conserved and may be the cause of inefficient splicing found specifically in human tissue. Our data correct GenomeScan annotation of this region of the human genome and describe functional gene discovery in mammals not recognized in human EST projects.

Amino Acid Sequence↗

PubMed2003

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human.

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3' end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14.

Algorithms↗