Unknown,Transcriptomics,Genomics,Proteomics

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Seeking novel mammalian genes using transcript profiling and genotyping of a human radiation hybrid panel

ABSTRACT: Using a human-hamster radiation hybrid panel (G3), we have mapped loci regulating gene expression due to copy number increase. Copy number from radiation hybrid clones were measured relative to the A23 hamster recipient line. Expression from 79 radiation hybrid clones were measured in duplicate and compared to expression of the A23 hamster recipient line (GA_illumina_expression1 and GA_illumina_expression2). Expression of liver, kidney and heart from human and hamster are also included (GA_illumina_expression_R).

ORGANISM(S): Cricetulus griseus

SUBMITTER: Richard Wang

PROVIDER: E-GEOD-19003 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The heterotransplantation of human tumors to immune-deprived rodents has become an important preclinical tool for evaluating human tumor biology and responsiveness to therapeutic agents, based on the supposition that they reflect their original human origin. However, we demonstrate that such tumors can in fact change genetically, either by becoming tumors of the animal recipient or by becoming a hybrid of the human tumor and the animal hostâs cells. Three human tumors transplanted to the cheek pouch of golden hamsters resulted in permanently transplantable tumors that metastasize in the hamsters. Based on diverse lines of evidence, these transplants from a human glioblastoma multiforme and two Hodgkin lymphomas were malignant hybrids, showing both human and hamster genes in the malignant tumor cells, while retaining the morphology of the original human tumors. We demonstrate that even after being preserved for over 40 years in paraffin blocks, microarray analysis of RNA from these transplants disclosed over 3000 human genes derived from all 23 human chromosome pairs amongst these tumor transplants. A total of 3759 probe sets (ranging from 1040 to 1303 in each transplant) detected human gene transcripts in formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors stored for over 40 years; the probe sets unambiguously map to 3107 unique Human Entrez Gene IDs and are representative of all human chromosomes, although, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing transcripts from 33 human genes , were detected in all hybrid tumor samples. Five of these 33 genes encode transcription factors that regulate cell growth and differentiation, five encode cell adhesion and transmigration-associated proteins known to participate in oncogenesis and/or metastasis and invasion, and additional genes encode components of pathways involved with signal transduction, regulation of apoptosis, DNA repair, and multidrug resistance. We posit that in-vivo fusion may disclose genes implicated in tumor progression, as well as gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other functions of the donor DNA. Human gene expression profiles were determined by microarray analysis for 3 different Human-Hamster hybrid tumors, two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749), that were first generated in the hamster cheek pouch after human tumor grafting and then propagated in hamsters and in cell cultures for years. Human gene expression was assessed using total RNA isolated from FFPE samples from GW-532 generations 2 and 34, GW-584 generation 28, and GB-749, in comparison to that for a Hamster control RNA sample isolated from a Hamster melanoma cell line (ATCC CCL-49). Expressed human genes were identified using MAS 5.0 signal expression values and detection P-values in the following manner: 1) Unannotated probe sets, as well as probe sets with no signal value greater than the median signal for AFFX spike-in controls with all Absent Detection Calls, were omitted from further analysis; 2) All remaining signal values for the hamster cell line sample were multiplied by the ratio of the median signal in all FFPE hybrid samples for AFFX spike-in control probe sets called present in all samples divided by the median signal for the same probe sets in the hamster CCL-49 sample; 3) Human transcripts were considered positive in a human-hamster hybrid FFPE sample if (a) a probe set signal exhibited a 2-fold or greater increase in any FFPE hybrid sample compared to the CCL-49 coontrol sample, (b) the fold change was greater than 2 standard deviations for that probe set across the FFPE samples, and (c) was called present (P) or marginal (M) for at least one or more FFPE samples.

Project description:Fast-growing Eucalyptus grandis trees are one of the most efficient producers of wood in South Africa. It is essential to maximize the effectiveness of these plantations by increasing their productivity, the quality and value of their products. We used microarray-based DNA-amplified fragment length polymorphism (AFLP) analysis in combination with expression profiling to develop fingerprints and profile gene expression of wood-forming tissue of seven individual E. grandis trees. A 1532-probe cDNA microarray was constructed by arraying 768 cDNA-AFLP fragments and 810 cDNA library clones from seven individual E. grandis trees onto silanised slides. The results revealed that 32% of the spotted fragments showed distinct expression patterns (with a fold change of at least 1.4 or -1.4 and a p value of 0.01) and could be grouped into clusters representing co-expressed genes. Evaluation of the binary distribution of cDNA-AFLP fragments on the array showed that the individual genotypes could be discriminated. A simple, yet general method was developed for genotyping and expression profiling of wood-forming tissue of E. grandis trees differing in their splitting characteristics and in their lignin contents. Evaluation of gene expression profiles and the binary distribution of cDNA-AFLP fragments on the chip suggest that the prototype chip developed could be useful for transcript profiling and for the identification of Eucalyptus trees with preferred wood quality traits in commercial breeding programmes. Transcriptional profiling and genotyping of seven E. grandis trees , namely (741-H (high lignin), 108-L (low lignin), 243-L (low lignin), 1/23/4-HS (high splitting), 1/71/6-HS (high splitting), 1/91/7-LS (low splitting) and 1/92/7-LS (low splitting), were performed with an in-house spotted cDNA microarray. All trees were characterized for their splitting qualities and lignin content and only the trees best corresponding to the selected traits were used for further analysis (Verryn and Turner 2000). RNA was isolated and cDNA synthesis was performed. A reference design microarray experiment was conducted and tree 1/23/4-HS served as a reference. Samples were fluorescently labelled with Cy5 or Cy3 dye and co-hybridized overnight. Two biological and one technical replicate (using independent labelling reactions) was performed, each replication consisting of a reverse labelling experiment.

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Seeking novel mammalian genes using transcript profiling and genotyping of a human radiation hybrid panel

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