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Mining the transcriptome - methods and applications
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-3811-5439
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods.

The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal.

This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype.

To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank.

Place, publisher, year, edition, pages
Stockholm: KTH , 2006. , 62 p.
Series
Theses in philosophy from the Royal Institute of Technology, ISSN 1650-8831
Keyword [en]
transcriptome, gene expression profiling, EST, microarray, RNA amplification, stem cells, neurosphere
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4115ISBN: 91-7178-436-5 (print)OAI: oai:DiVA.org:kth-4115DiVA: diva2:10803
Public defence
2006-10-13, FR4, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100927Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2010-09-27Bibliographically approved
List of papers
1. Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana
Open this publication in new window or tab >>Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana
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2005 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 5, 5- p.Article in journal (Refereed) Published
Abstract [en]

Background: Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags ( ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific.

Results: We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic ( auxin) treatment. A total of 270 genes were identified as differentially expressed ( 120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes.

Conclusions: The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21 000 Arabidopsis transcripts.

Keyword
Arrays, Genes, Image processing, Nucleic acids, Optimization, Probes, Purification
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-6165 (URN)10.1186/1472-6750-5-5 (DOI)000227244000001 ()2-s2.0-26844497995 (Scopus ID)
Note
QC 20100831Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2017-12-14Bibliographically approved
2. Catalog of gene expression in adult neural stem cells and their in vivo microenvironment
Open this publication in new window or tab >>Catalog of gene expression in adult neural stem cells and their in vivo microenvironment
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2006 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 312, no 10, 1798-1812 p.Article in journal (Refereed) Published
Abstract [en]

 Stem cells generally reside in a stem cell micro environment, where cues for self-renewal and differentiation are present. However, the genetic program underlying stem cell proliferation and multipotency is poorly understood. Transcriptome analysis of stem cells and their in vivo microenvironment is one way of uncovering the unique sternness properties and provides a framework for the elucidation of stem cell function. Here, we characterize the gene expression profile of the in vivo neural stem cell microenvironment in the lateral ventricle wall of adult mouse brain and of in vitro proliferating neural stem cells. We have also analyzed an Lhx2-expressing hematopoietic-stem-cell-like cell line in order to define the transcriptome of a well-characterized and pure cell population with stem cell characteristics. We report the generation, assembly and annotation of 50,792 high-quality 5'-end expressed sequence tag sequences. We further describe a shared expression of 1065 transcripts by all three stem cell libraries and a large overlap with previously published gene expression signatures for neural stem/progenitor cells and other multipotent stem cells. The sequences and cDNA clones obtained within this framework provide a comprehensive resource for the analysis of genes in adult stem cells that can accelerate future stem cell research.

Keyword
Digital signature, EST, Gene expression, Lateral ventricle wall, Microenvironment, Neural stem cell, Neurosphere, Transcriptome
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-6166 (URN)10.1016/j.yexcr.2006.02.012 (DOI)000238179100010 ()2-s2.0-33646714932 (Scopus ID)
Note
QC 20100902Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2017-12-14Bibliographically approved
3. Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method
Open this publication in new window or tab >>Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method
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2005 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 6, no 28, 13- p.Article in journal (Refereed) Published
Abstract [en]

Background: Neural stem cells ( NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression.

Results: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability.

Conclusions: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.

Keyword
central-nervous-system, adult human brain, microarray data, differentiation, neurospheres, neurons, expression, generation, single
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6167 (URN)10.1186/1471-2202-6-28 (DOI)000229042300001 ()2-s2.0-26444571449 (Scopus ID)
Note
QC 20100927Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2017-12-14Bibliographically approved
4. Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation
Open this publication in new window or tab >>Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation
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2005 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 6, 55- p.Article in journal (Refereed) Published
Abstract [en]

Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.

Results: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP.

Conclusion: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.

Keyword
CENTRAL-NERVOUS-SYSTEM, ADULT-MOUSE BRAIN, PARKINSONS-DISEASE, MICROARRAY DATA, DENTATE GYRUS, DIFFERENTIATION, TRANSPLANTATION, AMPLIFICATION, PROGENITORS, NEURONS
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6168 (URN)10.1186/1471-2202-6-55 (DOI)000231880900001 ()2-s2.0-26444590557 (Scopus ID)
Note
QC 20100914Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2017-12-14Bibliographically approved
5. p53 suppresses the self-renewal of adult neural stem cells
Open this publication in new window or tab >>p53 suppresses the self-renewal of adult neural stem cells
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2006 (English)In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 133, no 2, 363-369 p.Article in journal (Refereed) Published
Abstract [en]

There is increasing evidence that tumors are heterogeneous and that a subset of cells act as cancer stem cells. Several proto-oncogenes and tumor suppressors control key aspects of stem cell function, suggesting that similar mechanisms control normal and cancer stem cell properties. We show here that the prototypical tumor suppressor p53, which plays an important role in brain tumor initiation and growth, is expressed in the neural stem cell lineage in the adult brain. p53 negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analysis of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregulation of p21 expression. These data implicate p53 as a suppressor of tissue and cancer stem cell self-renewal.

Keyword
Adult, Cancer, Microarray data, Mouse, p21 (Cdkn1a), p53, Self-renewal, Stem cell, Trp53
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6169 (URN)10.1242/dev.02208 (DOI)000235345700017 ()2-s2.0-32244436592 (Scopus ID)
Note
QC 20100927Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2017-12-14Bibliographically approved
6. Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
Open this publication in new window or tab >>Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
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2006 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 7, 75- p.Article in journal (Refereed) Published
Abstract [en]

Background: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.

Results: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.

Conclusion: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.

Keyword
LIM-HOMEOBOX GENE, SEPTUM TRANSVERSUM MESENCHYME, TRANSCRIPTION FACTORS, MICROARRAY DATA, SELF-RENEWAL, MOUSE EMBRYO, IN-VITRO, LIVER, IDENTIFICATION, MULTIPOTENT
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-6170 (URN)10.1186/1471-2164-7-5 (DOI)000237424900001 ()2-s2.0-33646691980 (Scopus ID)
Note

QC 20100916

Available from: 2006-09-22 Created: 2006-09-22 Last updated: 2017-12-14Bibliographically approved

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