Anette Rink

Rearranged gene order between pig and human in a QTL region on SSC 7

On porcine Chromosome 7, the region surrounding the MHC region contains QTL influencing many traits including growth, back fat thickness, and carcass composition. Towards the identification of the responsible gene(s), this article describes an increase of density of the radiated hybrid map of SSC 7 in the q11-q14 region and the comparative analysis of gene order on the porcine RH map and human genome assembly. Adding 24 new genes in this region, we were able to build a framework map that fills in gaps on the previous maps. The new software Carthagene was used to build a robust framework in this region. Comparative analysis of human and porcine maps revealed a global conservation of gene order and of distances between genes. A rearranged fragment of around 3.7 Mb was, however, found in the pig approximately 20 Mb upstream from the expected location on the basis of the human map. This rearrangement, found by RH mapping on the IMpRH 7.000 rads panel, has been confirmed by two-color FISH and by mapping on the high resolution IMNpRH2 12.000 rads panel. The rearranged fragment contains two microsatellites found at the most likely QTL location in the INRA QTL experiment. It also contains the BMP5 gene, which, together with CLPS, could be considered as a possible candidate.

Radiation hybrid map of the porcine genome comprising 2035 EST loci

The IMpRH7000-rad radiation hybrid panel was used to map 2035 expressed sequence tags (ESTs) at a minimum LOD score of 4.0. A total of 134 linkage groups covers 57,192 cR or 78% of the predicted size of the porcine and 71% of the human genome, respectively. Approximately 81% (1649) of the porcine ESTs were annotated against the NCBI nonredundant database; 1422 mapped in silico to a location in build 35.1 of the human genome sequence (HGS) and 1185 to a gene and location in build 35.1 HGS. The map revealed 40 major breaks in synteny (1.00e−25 and lower) with the human genome, 37 of which fall within a single chromosome. At this improved level of resolution and coverage, porcine chromosomes (SSC) 2, 5, 6, 7, 12, and 14 remain “gene-rich” and homologous to human chromosomes (HSA) 17, 19, and 22, while SSC 1, 8, 11, and X have been confirmed to correspond to the “gene-deserts” on HSA 18, 4, 13, and X.

Normalized cDNA libraries from a porcine model of orthopedic implant-associated infection

Abstract. Staphylococcal infections that result from an alteration in a patients immune response at the surgical site are a major problem in procedures that incorporate biomaterials in trauma surgery and joint replacement. Diagnosis of infection based on pathogen detection is difficult and exacerbated by increasing numbers of partially or totally resistant strains of nosocomial pathogens, particularly Staphylococcus aureus. Expression profiling of a hosts cellular immune response could facilitate the identification of the pathways involved in pathogen recognition and eradication and could lead to more rational design of drugs and therapies. To this end, we constructed and characterized ten individually tagged and directionally cloned cDNA libraries from peripheral blood cells (PBC), spleen (Sp), thymus (Th), lymph node (LN), and bone marrow (BM) from immunologically naive and challenged pigs as part of an implant-associated orthopedic model of deep infection. Three of these libraries were normalized at C0t values 5, 10, 20, and 30. The libraries comprise more than 20 million primary transformants with an average insert length >1.4 kb. Cluster analysis of 7620 ESTs revealed 1029 clusters containing an average of 3.6 sequences and 3846 singletons. Gene discovery is estimated to be ∼64%. Searches of public databases resulted in 49.3% annotated porcine sequences, of which 22.2% had significant homologies to ESTs from a variety of species, and 28.5% were without a significant match in any public database. We also identified 9.1% ESTs as involved in host cell and organism defense and 11.5% related to cell signaling and communication. These sequences, together with the 28.5% appearing as novel, are of specific interest to the infectious disease process.

cDNA Microarray Analysis of Host-Pathogen Interactions in a Porcine In Vitro Model for Toxoplasma gondii Infection

ABSTRACT Toxoplasma gondii induces the expression of proinflammatory cytokines, reorganizes organelles, scavenges nutrients, and inhibits apoptosis in infected host cells. We used a cDNA microarray of 420 annotated porcine expressed sequence tags to analyze the molecular basis of these changes at eight time points over a 72-hour period in porcine kidney epithelial (PK13) cells infected with T. gondii. A total of 401 genes with Cy3 and Cy5 spot intensities of ≥500 were selected for analysis, of which 263 (65.6%) were induced ≥2-fold (expression ratio, ≥2.0; P ≤ 0.05 [t test]) over at least one time point and 48 (12%) were significantly down-regulated. At least 12 functional categories of genes were modulated (up- or down-regulated) by T. gondii. The majority of induced genes were clustered as transcription, signal transduction, host immune response, nutrient metabolism, and apoptosis related. The expression of selected genes altered by T. gondii was validated by quantitative real-time reverse transcription-PCR. These results suggest that significant changes in gene expression occur in response to T. gondii infection in PK13 cells, facilitating further analysis of host-pathogen interactions in toxoplasmosis in a secondary host.

Differential gene expression analysis during porcine hepatocyte spheroid formation

Primary porcine hepatocytes cultured in suspension self-assemble into multicellular aggregates or spheroids that display enhanced liver-specific functional capability and remain viable for an extended period of time in vitro. The molecular events underlying the process of spheroid formation were explored by differential gene expression analysis. Critical time points in spheroid formation were first identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of stress-related gene expression levels at different stages of spheroid formation. Suppression subtractive hybridization was used to identify transcripts up- or down-regulated at different stages of spheroid formation. Subsequently, three sets of reciprocal subtractions, comparing freshly isolated hepatocytes, spheroid-forming hepatocytes, and mature spheroids were carried out, and differentially expressed transcripts were isolated, cloned, sequenced, and annotated. A total of 65 genes and 14 novel transcripts were identified as differentially expressed, and very high sequence conservation between pig and human transcripts was observed. The resultant expressed sequence tags (ESTs) revealed a rapid decrease in the transcript levels of a subset of liver-specific genes, cytochrome P450s, and enzymes involved in heme biosynthesis, as well as up-regulation of genes involved in calcium-dependent vesicle trafficking and a number of acute-phase proteins in mature spheroids. Previous morphological and functional data on hepatocyte spheroid formation support cellular polarization of the hepatocyte into apical and basolateral domains in spheroids. This is important for the re-emergence of differentiated functions in vitro and is reflected by differences in gene expression patterns.

A Mathematical Model for Suppression Subtractive Hybridization

Suppression subtractive hybridization (SSH) is frequently used to unearth differentially expressed genes on a whole-genome scale. Its versatility is based on combining cDNA library subtraction and normalization, which allows the isolation of sequences of varying degrees of abundance and differential expression. SSH is a complex process with many adjustable parameters that affect the outcome of gene isolation.We present a mathematical model of SSH based on DNA hybridization kinetics for assessing the effect of various parameters to facilitate its optimization. We derive an equation for the probability that a particular differentially expressed species is successfully isolated and use this to quantify the effect of the following parameters related to the cDNA sample: (a) mRNA abundance; (b) partial sequence complementarity to other species; and (3) degree of differential expression. We also evaluate the effect of parameters related to the process, including: (a) reaction times; and (b) extent of driver excess used in the two hybridization reactions. The optimum set of process parameters for successful isolation of differentially expressed species depends on transcript abundance. We show that the reaction conditions have a significant effect on the occurrence of false-positives and formulate strategies to isolate specific subsets of differentially expressed genes. We also quantify the effect of non-specific hybridization on the false-positive results and present strategies for spiking cDNA sequences to address this problem.

Genomic exploration on Chinese hamster ovary cells

Industrially important recombinant CHO cell clones carry many desirable phenotypic traits that differentiate them from the parental clone. The molecular basis for these traits at the genomic level is not well understood. A better understanding of key genetic alterations related to these traits will greatly facilitate the development of new cell lines and new processes. To advance this goal, we are constructing a CHO cDNA microarray. A phage library was constructed using mRNA pooled from different CHO clones under different cultivation conditions. Over four thousand sequences have been randomly isolated and sequenced. A cDNA microarray based on these sequences was constructed and used in gene expression studies. The differentially expressed genes identified by the CHO cDNA microarray were compared to those observed in cross-species hybridization to mouse cDNA microarrays. Sequence comparison with known mouse genes indicates only a moderate degree of homology. The results represent a significant step toward large-scale gene expression profiling for industrial mammalian cell culture processes.