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Article: High-throughput flow cytometry-based assay to identify apoptosis-inducing proteins.

Sauermann, Mamatha / Hahne, Florian / Schmidt, Christian / Majety, Meher / Rosenfelder, Heiko / Bechtel, Stephanie / Huber, Wolfgang / Poustka, Annemarie / Arlt, Dorit / Wiemann, Stefan

Journal of biomolecular screening

2007 Volume 12, Issue 4, Page(s) 510–520

Abstract: After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for ... ...

Abstract	After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers.
MeSH term(s)	Apoptosis/physiology ; Apoptosis Regulatory Proteins/analysis ; Apoptosis Regulatory Proteins/biosynthesis ; Caspase 3/analysis ; Caspase 3/biosynthesis ; Cell Line ; Flow Cytometry ; Humans ; Pilot Projects
Chemical Substances	Apoptosis Regulatory Proteins ; Caspase 3 (EC 3.4.22.-)
Language	English
Publishing date	2007-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Validation Studies
ZDB-ID	1433680-7
ISSN	1552-454X ; 1087-0571
ISSN (online)	1552-454X
ISSN	1087-0571
DOI	10.1177/1087057107301271
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Modeling and managing experimental data using FuGE.

Jones, Andrew R / Lister, Allyson L / Hermida, Leandro / Wilkinson, Peter / Eisenacher, Martin / Belhajjame, Khalid / Gibson, Frank / Lord, Phil / Pocock, Matthew / Rosenfelder, Heiko / Santoyo-Lopez, Javier / Wipat, Anil / Paton, Norman W

Omics : a journal of integrative biology

2009 Volume 13, Issue 3, Page(s) 239–251

Abstract: The Functional Genomics Experiment data model (FuGE) has been developed to increase the consistency and efficiency of experimental data modeling in the life sciences, and it has been adopted by a number of high-profile standardization organizations. FuGE ...

Abstract	The Functional Genomics Experiment data model (FuGE) has been developed to increase the consistency and efficiency of experimental data modeling in the life sciences, and it has been adopted by a number of high-profile standardization organizations. FuGE can be used: (1) directly, whereby generic modeling constructs are used to represent concepts from specific experimental activities; or (2) as a framework within which method-specific models can be developed. FuGE is both rich and flexible, providing a considerable number of modeling constructs, which can be used in a range of different ways. However, such richness and flexibility also mean that modelers and application developers have choices to make when applying FuGE in a given context. This paper captures emerging best practice in the use of FuGE in the light of the experience of several groups by: (1) proposing guidelines for the use and extension of the FuGE data model; (2) presenting design patterns that reflect recurring requirements in experimental data modeling; and (3) describing a community software tool kit (STK) that supports application development using FuGE. We anticipate that these guidelines will encourage consistent usage of FuGE, and as such, will contribute to the development of convergent data standards in omics research.
MeSH term(s)	Computational Biology/methods ; Computer Simulation ; Flow Cytometry/instrumentation ; Flow Cytometry/methods ; Genomics/methods ; Models, Theoretical ; Reproducibility of Results ; Software ; User-Computer Interface
Language	English
Publishing date	2009-05-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2030312-9
ISSN	1557-8100 ; 1536-2310
ISSN (online)	1557-8100
ISSN	1536-2310
DOI	10.1089/omi.2008.0080
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: The full-ORF clone resource of the German cDNA Consortium

Bechtel, Stephanie / Rosenfelder, Heiko / Duda, Anny / Peter Schmidt, Christian / Ernst, Ute / Wellenreuther, Ruth / Mehrle, Alexander / Schuster, Claudia / Bahr, Andre / Blöcker, Helmut / Heubner, Dagmar / Hoerlein, Andreas / Michel, Guenter / Wedler, Holger / Köhrer, Karl / Ottenwälder, Birgit / Poustka, Annemarie / Wiemann, Stefan / Schupp, Ingo

2015

Abstract: Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus ... ...

Abstract	Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.
Subject code	572
Language	English
Publishing date	2015-09-04T08:28:36Z
Publishing country	de
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The full-ORF clone resource of the German cDNA Consortium

2015

Abstract	Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.
Subject code	572
Language	English
Publishing date	2015-09-04T08:28:36Z
Publishing country	de
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The full-ORF clone resource of the German cDNA Consortium

Michel Guenter / Hoerlein Andreas / Heubner Dagmar / Blöcker Helmut / Bahr Andre / Schuster Claudia / Mehrle Alexander / Wellenreuther Ruth / Ernst Ute / Schmidt Christian / Duda Anny / Rosenfelder Heiko / Bechtel Stephanie / Wedler Holger / Köhrer Karl / Ottenwälder Birgit / Poustka Annemarie / Wiemann Stefan / Schupp Ingo

BMC Genomics, Vol 8, Iss 1, p

2007 Volume 399

Abstract	Abstract Background With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.
Keywords	Genetics ; QH426-470 ; Biology (General) ; QH301-705.5 ; Science ; Q ; DOAJ:Genetics ; DOAJ:Biology ; DOAJ:Biology and Life Sciences ; Biotechnology ; TP248.13-248.65
Subject code	572
Language	English
Publishing date	2007-10-01T00:00:00Z
Publisher	BioMed Central
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: The LIFEdb database in 2006.

Mehrle, Alexander / Rosenfelder, Heiko / Schupp, Ingo / del Val, Coral / Arlt, Dorit / Hahne, Florian / Bechtel, Stephanie / Simpson, Jeremy / Hofmann, Oliver / Hide, Winston / Glatting, Karl-Heinz / Huber, Wolfgang / Pepperkok, Rainer / Poustka, Annemarie / Wiemann, Stefan

Nucleic acids research

2006 Volume 34, Issue Database issue, Page(s) D415–8

Abstract: LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced ... ...

Abstract	LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.
MeSH term(s)	Cell Proliferation ; Computational Biology ; DNA, Complementary/chemistry ; Databases, Genetic ; Gene Expression ; Genes ; Genomics ; Internet ; Proteins/analysis ; Proteins/genetics ; Proteins/metabolism ; Recombinant Fusion Proteins/metabolism ; Systems Integration ; User-Computer Interface
Chemical Substances	DNA, Complementary ; Proteins ; Recombinant Fusion Proteins
Language	English
Publishing date	2006-01-01
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2205588-5
ISSN	1362-4962 ; 1746-8272 ; 0305-1048 ; 0261-3166
ISSN (online)	1362-4962 ; 1746-8272
ISSN	0305-1048 ; 0261-3166
DOI	10.1093/nar/gkj139
Database	MEDical Literature Analysis and Retrieval System OnLINE

2006

Abstract	LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.
Keywords	Internet ; bioinformatics ; cell proliferation ; complementary DNA ; databases ; gene overexpression ; genes ; genomics ; proteins ; screening ; user interface
Language	English
Dates of publication	2006-0101
Size	p. D415-D418.
Document type	Article
ZDB-ID	186809-3
ISSN	0301-5610 ; 0305-1048
ISSN	0301-5610 ; 0305-1048
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: The full-ORF clone resource of the German cDNA Consortium.

Bechtel, Stephanie / Rosenfelder, Heiko / Duda, Anny / Schmidt, Christian Peter / Ernst, Ute / Wellenreuther, Ruth / Mehrle, Alexander / Schuster, Claudia / Bahr, Andre / Blöcker, Helmut / Heubner, Dagmar / Hoerlein, Andreas / Michel, Guenter / Wedler, Holger / Köhrer, Karl / Ottenwälder, Birgit / Poustka, Annemarie / Wiemann, Stefan / Schupp, Ingo

BMC genomics

2007 Volume 8, Page(s) 399

Abstract: Background: With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far ... ...

Abstract	Background: With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Results: Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion: The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.
MeSH term(s)	Cloning, Molecular/methods ; Codon, Terminator/genetics ; Computer Simulation ; Cooperative Behavior ; DNA Primers ; DNA, Complementary/genetics ; DNA-Directed DNA Polymerase/metabolism ; Databases, Genetic ; Genome, Human ; Germany ; Humans ; Models, Biological ; Open Reading Frames/genetics ; Polymerase Chain Reaction ; Quality Control ; Recombination, Genetic/genetics ; Sequence Analysis, DNA/methods ; User-Computer Interface
Chemical Substances	Codon, Terminator ; DNA Primers ; DNA, Complementary ; DNA-Directed DNA Polymerase (EC 2.7.7.7)
Language	English
Publishing date	2007-10-31
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1471-2164
ISSN (online)	1471-2164
DOI	10.1186/1471-2164-8-399
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Functional profiling: from microarrays via cell-based assays to novel tumor relevant modulators of the cell cycle.

Cancer research

2005 Volume 65, Issue 17, Page(s) 7733–7742

Abstract: Cancer transcription microarray studies commonly deliver long lists of "candidate" genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, ... ...

Abstract	Cancer transcription microarray studies commonly deliver long lists of "candidate" genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, is established as they were identified in large-scale genomics approaches. Strategies and tools are thus needed to distinguish genes and proteins with mere tumor association from those causally related to cancer. Here, we describe a functional profiling approach, where we analyzed 103 previously uncharacterized genes in cancer relevant assays that probed their effects on DNA replication (cell proliferation). The genes had previously been identified as differentially expressed in genome-wide microarray studies of tumors. Using an automated high-throughput assay with single-cell resolution, we discovered seven activators and nine repressors of DNA replication. These were further characterized for effects on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (G1-S transition) and anchorage-independent growth (tumorigenicity). One activator and one inhibitor protein of ERK1/2 activation and three repressors of anchorage-independent growth were identified. Data from tumor and functional profiling make these proteins novel prime candidates for further in-depth study of their roles in cancer development and progression. We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies.
MeSH term(s)	Animals ; Cell Cycle/genetics ; DNA Replication ; Gene Expression Profiling/methods ; Genes, cdc ; Humans ; MAP Kinase Signaling System/genetics ; Mice ; NIH 3T3 Cells ; Neoplasms/genetics ; Neoplasms/metabolism ; Neoplasms/pathology ; Oligonucleotide Array Sequence Analysis/methods ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics
Chemical Substances	RNA, Messenger
Language	English
Publishing date	2005-09-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1432-1
ISSN	1538-7445 ; 0008-5472
ISSN (online)	1538-7445
ISSN	0008-5472
DOI	10.1158/0008-5472.CAN-05-0642
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project.

Taylor, Chris F / Field, Dawn / Sansone, Susanna-Assunta / Aerts, Jan / Apweiler, Rolf / Ashburner, Michael / Ball, Catherine A / Binz, Pierre-Alain / Bogue, Molly / Booth, Tim / Brazma, Alvis / Brinkman, Ryan R / Michael Clark, Adam / Deutsch, Eric W / Fiehn, Oliver / Fostel, Jennifer / Ghazal, Peter / Gibson, Frank / Gray, Tanya /

Grimes, Graeme / Hancock, John M / Hardy, Nigel W / Hermjakob, Henning / Julian, Randall K / Kane, Matthew / Kettner, Carsten / Kinsinger, Christopher / Kolker, Eugene / Kuiper, Martin / Le Novère, Nicolas / Leebens-Mack, Jim / Lewis, Suzanna E / Lord, Phillip / Mallon, Ann-Marie / Marthandan, Nishanth / Masuya, Hiroshi / McNally, Ruth / Mehrle, Alexander / Morrison, Norman / Orchard, Sandra / Quackenbush, John / Reecy, James M / Robertson, Donald G / Rocca-Serra, Philippe / Rodriguez, Henry / Rosenfelder, Heiko / Santoyo-Lopez, Javier / Scheuermann, Richard H / Schober, Daniel / Smith, Barry / Snape, Jason / Stoeckert, Christian J / Tipton, Keith / Sterk, Peter / Untergasser, Andreas / Vandesompele, Jo / Wiemann, Stefan

Nature biotechnology

2008 Volume 26, Issue 8, Page(s) 889–896

MeSH term(s)	Databases as Topic ; Guidelines as Topic ; Information Dissemination ; Internet ; Oligonucleotide Array Sequence Analysis/standards ; Proteomics/standards ; Research Design/standards ; Vocabulary, Controlled
Language	English
Publishing date	2008-06-27
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/nbt.1411
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