shRNAs were assessed for off-target effects by comparing the gene expression profiles of cells that they had been infected into. shRNAs designed with the shERWOOD algorithm and house in the ultramir microRNA scafold were found to have very little off targeting. Overall design: Purpose: A major detriment to RNAi is off-targeting. We wished to assess the level of off targeting of microRNA (ultramiR) housed shERWOOD shRNAs as compared to similar shRNAs in the TRC collection. Methods: 5 shRNAs targeting each of two genes were infected into the 4T1 cell line. For each gene one shRNA was selected from the TRC collection and one based on the shERWOOD algorithm. For each gene, the exrpession profiles of the corresponding shRNA infected cells were compared using RNAseq. Conclusions: Highly similar profiles were observed between shERWOOD selected shRNAs. TRC shRNAs produced profiles indicative of off-targeting.
A computational algorithm to predict shRNA potency.
No sample metadata fields
View SamplesWe used Kras/Hras/Nras-triple knockout MEFs expressing recombinant Nras to test the off-target effect of 2 Kras siRNAs at different transfection concentrations.
Development of siRNA payloads to target KRAS-mutant cancer.
Specimen part
View SamplesWe analyzed miRNA-based shRNA off-target effects by transducing Trp53-/- MEFs at single- and high-copy with six well-characterized, potent and weak Trp53 shRNAs.
Development of siRNA payloads to target KRAS-mutant cancer.
Specimen part
View SamplesThis SuperSeries is composed of the SubSeries listed below.
An integrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance.
Specimen part, Treatment
View SamplesTo explore oncogene addiction programs in a genetically defined leukemia context we developed an AML mouse model driven by a conditional MLL-AF9 allele together with oncogenic Ras, which enabled us to examine the consequences of MLL-AF9 inhibition in established disease. In order to produce a tightly regulated system that was easy to monitor, we constructed two retroviral vectors containing dsRed-linked MLL-AF9 under control of a tetracycline response element promoter, and KrasG12D or NrasG12D linked to the Tet-off tet-transactivator, which activates TRE expression in a doxycycline repressible manner. Leukemias were generated by retroviral cotransduction of both vectors into hematopoietic stem and progenitor cells, which were transplanted into syngeneic mice. Cells harboring both constructs induced aggressive myelomonocytic leukemia. Five independent primary leukemia cell lines were established from bone marrow of terminal mice. Treatment of these lines with doxycycline rapidly turned off MLL-AF9 expression, and induced terminal myeloid differentiation and complete disease remission in vivo.
An integrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance.
Specimen part, Treatment
View SamplesUsing an integrative approach combining a Tet-off conditional AML mouse model, global expression profiling following suppression of the driving MLL-AF9 oncogene, and a new Tet-on conditional shRNA expression system we have identified Myb as critical mediator of addiction to MLL-AF9. Suppression of Myb in established AML in vivo terminates aberrant self-renewal and triggers a terminal myeloid differentiation program that precisely phenocopies the effects of suppressing MLL-AF9. Remarkably, suppressing Myb effectively eradicates aggressive and chemotherapy resistant AML.
An integrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance.
Specimen part
View SamplesTwo sets of wheat lines near-isogenic to Lr34 were used to compare gene expression profiles of wheat: 1. with and without Lr34 gene; 2. rust and mock inoculation; 3. distal and basal portion of the flag leaves. The two sets of wheat near-isogenic lines were used to subtract genetic background variations and to enrich Lr34-regulated gene expression profiles. The study is aimed to better understand the mechanisms of the well-known durable leaf rust resistance gene, Lr34, mediated resistance at the transcriptome level.
Gene expression patterns in near isogenic lines for wheat rust resistance gene lr34/yr18.
No sample metadata fields
View SamplesThe H4K16 acetyltransferase MOF plays a crucial role in dosage compensation in Drosophila, but has additional, global functions in gene control. We compared the molecular context and effect of MOF activity in male and female flies combining chromosome-wide mapping and transcriptome studies with analyses of defined reporter loci in transgenic flies. MOF distributes dynamically between two types of complexes, the Dosage Compensation Complex (DCC) and complexes containing MBD-R2, a global facilitator of transcription. These different targeting principles define the distribution of MOF between the X chromosome and autosomes and at transcription units with 5 or 3 enrichment.
The activation potential of MOF is constrained for dosage compensation.
Cell line
View SamplesThis SuperSeries is composed of the SubSeries listed below.
The MOF-containing NSL complex associates globally with housekeeping genes, but activates only a defined subset.
Sex, Specimen part, Cell line
View SamplesThe MOF-containing NSL complex binds to many but not all promoters of active genes and potentially contributes to their proper gene expression. It is currently unknown what determines whether an active gene is bound or not. Here, we provide evidence that the NSL complex primarily targets active promoters of most housekeeping genes. There, it co-localizes with the chromatin remodeler NURF and the histone methyltransferase Trithorax. Moreover, despite binding to most housekeeping genes, the NSL complex regulates only a subset of them, which are depleted for certain insulator binding-proteins and enriched for the core promoter motif Ohler 5. We suggest that the combination of general chromatin factors and core promoter motifs is predictive for whether a housekeeping gene is transcriptionally regulated by the NSL complex.
The MOF-containing NSL complex associates globally with housekeeping genes, but activates only a defined subset.
Cell line
View Samples