Development and utilization of a custom PCR array workflow: analysis of gene expression in mycoplasma genitalium and guinea pig (Cavia porcellus).
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ABSTRACT:
SUBMITTER: Veselenak RL
PROVIDER: S-EPMC4298676 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In science, the guinea pig is known as one of the gold standards for modeling human disease. It is especially important as a molecular and cellular biology model for studying the human immune system, as its immunological genes are more similar to human genes than are those of mice. The utility of the guinea pig as a model organism can be further enhanced by further characterization of the genes encoding components of the immune system. Here, we report the genomic organization of the guinea pig immunoglobulin (Ig) heavy and light chain genes. The guinea pig IgH locus is located in genomic scaffolds 54 and 75, and spans approximately 6,480 kb. 507 V(H) segments (94 potentially functional genes and 413 pseudogenes), 41 D(H) segments, six J(H) segments, four constant region genes (?, ?, ?, and ?), and one reverse ? remnant fragment were identified within the two scaffolds. Many V(H) pseudogenes were found within the guinea pig, and likely constituted a potential donor pool for gene conversion during evolution. The Ig? locus mapped to a 4,029 kb region of scaffold 37 and 24 is composed of 349 V(?) (111 potentially functional genes and 238 pseudogenes), three J(?) and one C(?) genes. The Ig? locus spans 1,642 kb in scaffold 4 and consists of 142 V(?) (58 potentially functional genes and 84 pseudogenes) and 11 J(?) -C(?) clusters. Phylogenetic analysis suggested the guinea pig's large germline V(H) gene segments appear to form limited gene families. Therefore, this species may generate antibody diversity via a gene conversion-like mechanism associated with its pseudogene reserves.
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Project description:Prolactin (PRL) is a multifunctional hormone with prominent roles in regulating growth and reproduction. The guinea pig (Cavia porcellus) has been extensively used in endocrine and reproduction research. Thus far, the PRL cDNA and protein have not been isolated from the guinea pig. In the present study, we used information derived from the public guinea pig genome database as a tool for identifying guinea pig PRL and PRL-related proteins. Guinea pig PRL exhibits prominent nucleotide and amino acid sequence differences when compared with PRLs of other eutherian mammals. In contrast, guinea pig GH is highly conserved. Expression of PRL and GH in the guinea pig is prominent in the anterior pituitary, similar to known expression patterns of PRL and GH for other species. Two additional guinea pig cDNAs were identified and termed PRL-related proteins (PRLRP1, PRLRP2). They exhibited a more distant relationship to PRL and their expression was restricted to the placenta. Recombinant guinea pig PRL protein was generated and shown to be biologically active in the PRL-responsive Nb2 lymphoma cell bioassay. In contrast, recombinant guinea pig PRLRP1 protein did not exhibit PRL-like bioactivity. In summary, we have developed a new set of research tools for investigating the biology of the PRL family in an important animal model, the guinea pig.
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Project description:The domesticated guinea pig, Cavia porcellus (Hystricomorpha, Rodentia), is an important laboratory species and a model for a number of human diseases. Nevertheless, genomic tools for this species are lacking; even its karyotype is poorly characterized. The guinea pig belongs to Hystricomorpha, a widespread and important group of rodents; so far the chromosomes of guinea pigs have not been compared with that of other hystricomorph species or with any other mammals. We generated full sets of chromosome-specific painting probes for the guinea pig by flow sorting and microdissection, and for the first time, mapped the chromosomal homologies between guinea pig and human by reciprocal chromosome painting. Our data demonstrate that the guinea pig karyotype has undergone extensive rearrangements: 78 synteny-conserved human autosomal segments were delimited in the guinea pig genome. The high rate of genome evolution in the guinea pig may explain why the HSA7/16 and HSA16/19 associations presumed ancestral for eutherians and the three syntenic associations (HSA1/10, 3/19, and 9/11) considered ancestral for rodents were not found in C. porcellus. The comparative chromosome map presented here is a starting point for further development of physical and genetic maps of the guinea pig as well as an aid for genome assembly assignment to specific chromosomes. Furthermore, the comparative mapping will allow a transfer of gene map data from other species. The probes developed here provide a genomic toolkit, which will make the guinea pig a key species to unravel the evolutionary biology of the Hystricomorph rodents.
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Project description:PurposeTo characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags.MethodsRNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS).ResultsCombined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.ConclusionsGenomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.
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Project description:The aim was to establish the genetic diversity and population structure of three guinea pig lines, from seven production zones located in Nariño, southwest Colombia. A total of 384 individuals were genotyped with six microsatellite markers. The measurement of intrapopulation diversity revealed allelic richness ranging from 3.0 to 6.56, and observed heterozygosity (Ho) from 0.33 to 0.60, with a deficit in heterozygous individuals. Although statistically significant (p < 0.05), genetic differentiation between population pairs was found to be low. Genetic distance, as well as clustering of guinea-pig lines and populations, coincided with the historical and geographical distribution of the populations. Likewise, high genetic identity between improved and native lines was established. An analysis of group probabilistic assignment revealed that each line should not be considered as a genetically homogeneous group. The findings corroborate the absorption of native genetic material into the improved line introduced into Colombia from Peru. It is necessary to establish conservation programs for native-line individuals in Nariño, and control genealogical and production records in order to reduce the inbreeding values in the populations.
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Project description:Background: The lack of adequate in vitro systems to isolate and propagate guinea pig adenovirus (GPAdV), a prevalent cause of respiratory illness of varaible severity in laboratory guinea pig colonies worldwide, has precluded its formal characterization to allow for the development of comprehensive diagnostic assays, and for the execution of complex pathogenesis and basic virology studies. Methods: Two strains of GPAdV were isolated in guinea pig ( Cavia porcellus) cell cultures from frozen archival infected animal tissue originated from colony outbreaks of pneumonia in Australia and the Czech Republic in 1996. Results: Commercially available guinea pig cell lines from colorectal carcinoma (GPC-16), fetal fibroblast (104-C1) and lung fibroblast (JH4 C1), and the tracheal epithelial cell line GPTEC-T developed in this study were able to support viral infection and early propagation. Sufficient viral DNA was recovered from cell cultures to PCR-amplify and obtain sequence data for the complete hexon gene and partial DNA polymerase and penton base genes. Phylogenetic analysis for the three regions of the genome provided strong evidence confirming GPAdV as a unique species in the genus Mastadenovirus. Conclusions: This study demonstrated the feasibility of propagating GPAdV in cultures of immortalized lines of GP cells of a variety of types, thus establishing a critical foundation for the development of a robust culture platform for virus stock production and titration. The generation and analysis of whole GPAdV genome sequences will provide additional data for a comprehensive description of the genetic organization of the viral genome and for a better assessment of genetic diversity between the two isolated strains.
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Project description:To manage pain effectively in people and animals, it is essential to recognise when pain is present and to assess its intensity. Currently there is very little information regarding the signs of post-surgical pain or its management in guinea pigs. Studies from other rodent species indicate that behaviour-based scoring systems can be used successfully to detect pain and evaluate analgesic efficacy. This preliminary study aimed to establish whether behaviour-based scoring systems could be developed to assess post-surgical pain in guinea pigs. This prospective, randomised, placebo-controlled study used 16 guinea pigs, and evaluated changes in behaviour following either anaesthesia alone or anaesthesia and orchiectomy. Behaviour was assessed using a combination of manual and automated scoring of remotely obtained video footage. A small number of behaviours were identified that appeared to have high specificity for pain caused by orchiectomy. However, the behaviours were displayed infrequently. The most common was a change in posture from standing to recumbency, sometimes with one hind leg extended either to the side or behind the body. A composite behaviour score incorporating these abnormal behaviours differentiated between the effects of surgery and anaesthesia alone (p<0.0001), and between animals that received analgesia post-operatively compared to an untreated group (p<0.0001). Although behavioural changes occurred in these guinea pigs after orchiectomy, the changes were relatively subtle and the individual specific pain-related behaviours occurred infrequently. However, it may prove possible to develop a behaviour-based scoring system for routine use in this species using a combination of pain-related behaviours.
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Project description:Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide with excessive incidence in low-and middle-income countries (LMIC). During a survey for C. jejuni from putative animal hosts in a town in the Peruvian Amazon, we were able to isolate and whole genome sequence two C. jejuni strains from domesticated guinea pigs (Cavia porcellus). The C. jejuni isolated from guinea pigs had a novel multilocus sequence type that shared some alleles with other C. jejuni collected from guinea pigs. Average nucleotide identity and phylogenetic analysis with a collection of C. jejuni subsp. jejuni and C. jejuni subsp. doylei suggest that the guinea pig isolates are distinct. Genomic comparisons demonstrated gene gain and loss that could be associated with guinea pig host specialization related to guinea pig diet, anatomy, and physiology including the deletion of genes involved with selenium metabolism, including genes encoding the selenocysteine insertion machinery and selenocysteine-containing proteins.
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Project description:Biological variation (BV) describes the physiological random fluctuation around a homeostatic set point, which is a characteristic of all blood measurands (analytes). That variation may impact the clinical relevance of the changes that are observed in the serial results for an individual. Biological variation is represented mathematically by the coefficient of variation (CV) and occurs within each individual (CVI) and between individuals in a population (CVG). Biological variation data can be used to assess whether population-based reference or subject-based reference intervals should be used for the interpretation of laboratory results through the calculation of the index of individuality (IoI). This study aimed to determine the biological variations, calculate the IoI and reference change values (RCV) of clinical chemistry analytes in an outbred strain colony of Hartley guinea pigs (GPs), and set the quality specifications for clinical chemistry analytes. Blood was collected from 16 healthy adult laboratory colony GPs via jugular venipuncture at weekly intervals over six weeks. All the samples were frozen and analyzed in a single run. Analytical, CVI, and CVG biological variations, together with the IoI and RCV, were calculated for each measurand. Based on the estimated BV, the calculated IoI was low for glucose, so individual reference intervals (RCV) should be used. The majority of the measurands should be interpreted using both population-based and subject-based reference intervals as the IoIs were intermediate.
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