Development of the first marmoset-specific DNA microarray (EUMAMA): a new genetic tool for large-scale expression profiling in a non-human primate

Contains fulltext : 34911.pdf (publisher's version ) (Open Access)BACKGROUND: The common marmoset monkey (Callithrix jacchus), a small non-endangered New World primate native to eastern Brazil, is becoming increasingly used as a non-human primate model in biomedical research, drug development and safety assessment. In contrast to the growing interest for the marmoset as an animal model, the molecular tools for genetic analysis are extremely limited. RESULTS: Here we report the development of the first marmoset-specific oligonucleotide microarray (EUMAMA) containing probe sets targeting 1541 different marmoset transcripts expressed in hippocampus. These 1541 transcripts represent a wide variety of different functional gene classes. Hybridisation of the marmoset microarray with labelled RNA from hippocampus, cortex and a panel of 7 different peripheral tissues resulted in high detection rates of 85% in the neuronal tissues and on average 70% in the non-neuronal tissues. The expression profiles of the 2 neuronal tissues, hippocampus and cortex, were highly similar, as indicated by a correlation coefficient of 0.96. Several transcripts with a tissue-specific pattern of expression were identified. Besides the marmoset microarray we have generated 3215 ESTs derived from marmoset hippocampus, which have been annotated and submitted to GenBank [GenBank: EF214838-EF215447, EH380242-EH382846]. CONCLUSION: We have generated the first marmoset-specific DNA microarray and demonstrated its use to characterise large-scale gene expression profiles of hippocampus but also of other neuronal and non-neuronal tissues. In addition, we have generated a large collection of ESTs of marmoset origin, which are now available in the public domain. These new tools will facilitate molecular genetic research into this non-human primate animal model

Effects of Aneuploidy on Genome Structure, Expression, and Interphase Organization in Arabidopsis thaliana

Aneuploidy refers to losses and/or gains of individual chromosomes from the normal chromosome set. The resulting gene dosage imbalance has a noticeable affect on the phenotype, as illustrated by aneuploid syndromes, including Down syndrome in humans, and by human solid tumor cells, which are highly aneuploid. Although the phenotypic manifestations of aneuploidy are usually apparent, information about the underlying alterations in structure, expression, and interphase organization of unbalanced chromosome sets is still sparse. Plants generally tolerate aneuploidy better than animals, and, through colchicine treatment and breeding strategies, it is possible to obtain inbred sibling plants with different numbers of chromosomes. This possibility, combined with the genetic and genomics tools available for Arabidopsis thaliana, provides a powerful means to assess systematically the molecular and cytological consequences of aberrant numbers of specific chromosomes. Here, we report on the generation of Arabidopsis plants in which chromosome 5 is present in triplicate. We compare the global transcript profiles of normal diploids and chromosome 5 trisomics, and assess genome integrity using array comparative genome hybridization. We use live cell imaging to determine the interphase 3D arrangement of transgene-encoded fluorescent tags on chromosome 5 in trisomic and triploid plants. The results indicate that trisomy 5 disrupts gene expression throughout the genome and supports the production and/or retention of truncated copies of chromosome 5. Although trisomy 5 does not grossly distort the interphase arrangement of fluorescent-tagged sites on chromosome 5, it may somewhat enhance associations between transgene alleles. Our analysis reveals the complex genomic changes that can occur in aneuploids and underscores the importance of using multiple experimental approaches to investigate how chromosome numerical changes condition abnormal phenotypes and progressive genome instability

Genomic approaches to unveil the physiological pathways activated in Arabidopsis treated with plant-derived raw extracts

DNA microarrays can be used to obtain a fingerprint of the transcriptional status of the plant or cell under a given condition and may be useful for characterising which genes respond, either by induction or repression, to novel stimuli or specific treatments. An in-depth bioinformatical analysis of all the data produced by microarrays can further highlight the metabolic or functional pathways most affected by the treatment. This approach has been used to investigate the effects induced by the treatment of different plant-derived raw materials, provided by Valagro SpA, on Arabidopsis seedlings. A clear example is represented by treatment with a raw plant-derived protein extract (VAL-P01). In this case the treatment induced genes related to ABA and osmotic stress treatment. We therefore demonstrated that VAL-P01 was able to mimic in planta the same pattern of responses linked to ABA treatment or osmotic stress, making the plant stronger against possible further stresses. Another plant extract, VAL-P02, was shown to be significantly altering the transcription of senescence genes, making it an ideal candidate adjuvant for the prolonged shelf-life of vegetal products

A specific group of genes respond to cold dehydration stress in cut Alstroemeria flowers whereas ambient dehydration stress accelerates developmental senescence expression patterns

Petal development and senescence entails a normally irreversible process. It starts with petal expansion and pigment production, and ends with nutrient remobilization and ultimately cell death. In many species this is accompanied by petal abscission. Post-harvest stress is an important factor in limiting petal longevity in cut flowers and accelerates some of the processes of senescence such as petal wilting and abscission. However, some of the effects of moderate stress in young flowers are reversible with appropriate treatments. Transcriptomic studies have shown that distinct gene sets are expressed during petal development and senescence. Despite this, the overlap in gene expression between developmental and stress-induced senescence in petals has not been fully investigated in any species. Here a custom-made cDNA microarray from Alstroemeria petals was used to investigate the overlap in gene expression between developmental changes (bud to first sign of senescence) and typical post-harvest stress treatments. Young flowers were stressed by cold or ambient temperatures without water followed by a recovery and rehydration period. Stressed flowers were still at the bud stage after stress treatments. Microarray analysis showed that ambient dehydration stress accelerates many of the changes in gene expression patterns that would normally occur during developmental senescence. However, a higher proportion of gene expression changes in response to cold stress were specific to this stimulus and not senescence related. The expression of 21 transcription factors was characterized, showing that overlapping sets of regulatory genes are activated during developmental senescence and by different stresses

Gene expression profiling in prepubertal and adult male mice using cDNA and oligonucleotide microarrays

Variations in gene expression are the basis of differences in cell and tissue function, response to DNA damaging agents, susceptibility to genetic disease, and cellular differentiation. The purpose of this dissertation research was to characterize variation in basal gene expression among adult mouse tissues for selected stress response, DNA repair and damage control genes and to utilize variation in temporal gene expression patterns to identify candidate genes associated with germ cell differentiation from mitosis through meiosis in the prepubertal mouse testis. To accomplish these goals, high throughput analyses of gene expression were performed using custom cDNA and random oligonucleotide microarrays. CDNA microarray technology was optimized by evaluating the effects of multiple hybridization and image analysis methodologies on the magnitude of background-subtracted hybridization signal intensities. The results showed that hybridizing lower probe quantities in a buffer developed at Lawrence Livermore National Laboratory to tryptone-blocked microarrays improved signal intensities. In addition, the error in expression ratio measurements was significantly reduced when microarray images were preprocessed. A custom cDNA microarray comprised of 417 genes and enriched for stress response, DNA repair, and damage control genes was used to investigate basal gene expression differences among adult mouse testis, brain, liver, spleen, and heart. Genes with functions related to stress response exhibited the most variation in expression among tissues whereas DNA repair-associated gene expression varied the least. Random oligonucleotide microarrays comprised of ∼10,000 genes were used to profile changes in gene expression during the first wave of spermatogenesis in the prepubertal mouse testis. Approximately 550 genes were differentially expressed as male germ cells differentiated from spermatogonia to primary spermatocytes. These findings suggest that the 313 unannotated sequences and 178 genes with known functions in other biological pathways have spermatogenesis-associated roles. This dissertation research showed that microarrays are a useful tool for quantitating the expression of large numbers of genes in parallel under normal physiological conditions and during differentiation. It has also provided candidate genes for future investigations of the molecular mechanisms underlying (1) tissue-specific DNA damage response and genetic disease susceptibility and (2) cellular differentiation during the onset and progression of spermatogenesis