NGFN-PLUS
Cellular Screening Systems
| Coordinator: | Dr. Ulrich Tschulena | |
| Institution: | DKFZ Heidelberg | |
| Homepage: | www.dkfz.de |
The major goal of this subproject is to provide expertise, capacities and technologies to identify and to determine the cellular function of disease-relevant genes and proteins. High-throughput functional cellular assays have been established and validated in NGFN-2 that will be complemented by novel assays. Cellular Screening Systems thus forms the backbone for high-throughput functional gene and protein analysis in NGFNplus.
Genes and proteins (~ 1,200) that have been pre-selected during NGFN-2 will be challenged for phenotypic changes that are induced by overexpression (ORFeome resource) and knock-down (RNAi), to define the set of candidate proteins that shall be further analysed and validated, to enhance knowledge on the cross-talk of growth-factor and hormone signalling and the impact on cell cycle and drug response.Within the functional profiling pipeline of the NGFN-2 SMP-Cell, the disease-oriented subprojects of this collaboration have developed and validated cellular assays that are applied in gene gain- and in gene loss-of-function screens at medium up to the whole-genome scale.
The ORFeome and RNAi resources of SMP-Cell are exploited there to identify novel proteins involved in cell cycle regulation, cell signalling, and other cancer-relevant processes.
The NGFN-2 SMP-Cell has its major disease focus on cancer.
Consequently, the majority of cellular functional assays that have been developed address cancer-relevant cellular processes:
Cell signalling: Erk1/2 and P38 assays have been carried out measuring the activation/phosphorylation level of the respective kinases in response to perturbations, to identify activators or inhibitors of the different pathways.
Cell cycle/cell growth: DNA replication assays measure the cell cycle status based on 7-AAD-DNA staining, and measure cell growth in general (WST-1).
Apoptosis: A Caspase-3 assay analyses the activation state of endogenous Caspase-3 as a marker for the induction of apoptosis.
Cell detachment and invasion: Cells in the supernatant or cells having passed an artificial basal lamina are counted, respectively, to measure phenotypic modulations induced by perturbations.
All these assays are all fully implemented and validated for their reproducibility, significance, and impact of results. Candidate genes identified in any primary screen will be validated in secondary screens with a complementary phenotypic read-out, and/or complementary perturbations.
Genes and proteins (~ 1,200) that have been pre-selected during NGFN-2 will be challenged for phenotypic changes that are induced by overexpression (ORFeome resource) and knock-down (RNAi), to define the set of candidate proteins that shall be further analysed and validated, to enhance knowledge on the cross-talk of growth-factor and hormone signalling and the impact on cell cycle and drug response.Within the functional profiling pipeline of the NGFN-2 SMP-Cell, the disease-oriented subprojects of this collaboration have developed and validated cellular assays that are applied in gene gain- and in gene loss-of-function screens at medium up to the whole-genome scale.
The ORFeome and RNAi resources of SMP-Cell are exploited there to identify novel proteins involved in cell cycle regulation, cell signalling, and other cancer-relevant processes.
The NGFN-2 SMP-Cell has its major disease focus on cancer.
Consequently, the majority of cellular functional assays that have been developed address cancer-relevant cellular processes:
Cell signalling: Erk1/2 and P38 assays have been carried out measuring the activation/phosphorylation level of the respective kinases in response to perturbations, to identify activators or inhibitors of the different pathways.
Cell cycle/cell growth: DNA replication assays measure the cell cycle status based on 7-AAD-DNA staining, and measure cell growth in general (WST-1).
Apoptosis: A Caspase-3 assay analyses the activation state of endogenous Caspase-3 as a marker for the induction of apoptosis.
Cell detachment and invasion: Cells in the supernatant or cells having passed an artificial basal lamina are counted, respectively, to measure phenotypic modulations induced by perturbations.
All these assays are all fully implemented and validated for their reproducibility, significance, and impact of results. Candidate genes identified in any primary screen will be validated in secondary screens with a complementary phenotypic read-out, and/or complementary perturbations.
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