|Quantity:||96 x 1ug transfection-ready DNA|
BioCat offers SwitchGear Genomics´panel of 88 transfection-ready reporter constructs to allow you to measure Hypoxia pathway activation. The SwitchGear Hypoxia promoter targets were picked from their genome-wide promoter collection based on sequence motif analysis, expression studies, and transcription factor binding data. SwitchGear conducted experiments to generate a profile across the set of constructs with and without Hypoxia activation. These data are summarized in the heatmap below.
The complete pathway profiling panel of constructs in a plate format allows you to characterize the effects of many conditions across the entire Hypoxia pathway.
A smaller biomarker subset of key responder constructs that show a strong induction response and can be used as biomarkers in screening applications, is also available, see link below.
Hypoxia (HIF1a) Profiling Plate Heatmap: The selected constructs represent the SwitchGear Hypoxia Profiling Plate. The heatmap summarizes the untreated activity and induction response for each construct in the pathway profiling plate using one common induction strategy. The chart summarizes the induction ratios for each construct in HT1080 human fibrosarcoma cells that were transfected with promoter-reporter constructs and were untreated or incubated at 1% O2, or treated with 100uM DFO for 24 hours. On the right side of the figure, the colors represent log2 transformed ratios of treated/untreated activity according to the color scale shown at the base of the heatmap. Black indicates no change, and intensity of red or blue indicates levels of induction or repression, respectively. On the left side, the intensity of yellow parallels the activity of the promoter in the unstimulated or untreated condition. All untreated signals are relative to the highest and lowest values within the individual experiment.
Eight Control Constructs included
The included SwitchGear Transfection Control Set can be used for normalization of your profiling data and to determine your conditions for maximum transfection efficiency and reproducibility.
The following negative and positive controls are provided:
• 4 constructs with random 1kb fragments from the human genome (R1, R2, R3, R4)
S790001-SW 5ug transfection-ready DNA of
LightSwitch GoClone Negative Control Random Sequence Promoter Reporter Construct (Random Genomic DNA from chr11+) (R01_PROM)
S790002-SW 5ug transfection-ready DNA of
LightSwitch GoClone Negative Control Random Sequence Promoter Reporter Construct (Random Genomic DNA from chr1+) (R02_PROM)
S790003-SW 5ug transfection-ready of
LightSwitch GoClone Negative Control Random Sequence Promoter Reporter Construct (Random Genomic DNA from chr6+) (R03_PROM)
S790004-SW 5ug transfection-ready of
LightSwitch GoClone Negative Control Random Sequence Promoter Reporter Construct (Random Genomic DNA from chr16+) (R04_PROM)
• 4 constructs from housekeeping genes with promoter activities of varying strengths (P1, P2, P3, P4)
S708908-SW 5ug transfection-ready DNA of
LightSwitch GoClone Positive Control Promoter Construct for Housekeeping Gene RPL10 (RPL10_PROM)
S717678-SW 5ug transfection-ready DNA of
LightSwitch GoClone Positive Control Promoter Construct for Housekeeping Gene ACTB (ACTB_PROM)
S721613-SW 5ug transfection-ready DNA of
LightSwitch GoClone Positive Control Promoter Construct for Housekeeping Gene LDHA (LDHA_PROM)
S721624-SW 5ug transfection-ready DNA of
LightSwitch GoClone Positive Control Promoter Construct for Housekeeping Gene GAPDH (GAPDH_PROM)
96 x 5 ug purified plasmid DNA
Concentration 30ng/ul each
Quality >1.75 260/280 ratio, sequence verified
LightSwitch GoClones should be used with LightSwitch Assay Reagents for maximum sensitivity, brightness, and dynamic range
Applications of SwitchGear Hypoxia Pathway Screening Sets:
Measure the activity of regulatory elements across the full Hypoxia-related pathway with transfection-ready promoter and 3′UTR assays
• Understand the mechanisms by which Hypoxia regulated genes are induced or repressed
• Quantify the functional consequences of transcription factor binding – while many other technologies give a qualitative view of transcription factor level, the SwitchGear promoter reporter assays quantify the effects of transcription factor binding
• Confirm data from ChIP-chip or ChIP-sequencing experiments
• Measure the effect of sequence variants and mutagenesis on promoter function
• Screen for promoter activation or develop activity profiles across the Hypoxia signaling pathway for many compounds or conditions in parallel
• SwitchGear also offers 3′UTR reporters for studying transcriptional stability, translational repression, and miRNA function. See link below.
Hypoxia, low oxygen concentration in cells, is a characteristic of various physiological processes such as the regulation of cell differentiation, blood vessel formation, and wound healing. Hypoxia is also widely associated with diseases such as anemia, diabetes, kidney disease, cardiovascular disease, hypertension, sickle cell disease, and many cancers. Cells under hypoxic stress activate a signaling cascade of hypoxia-responsive genes to aid the cell into either adapting to the low oxygen conditions or undergoing apoptosis. This response to restore cellular homeostasis is primarily initiated and regulated by the family of hypoxia inducible transcription factors (HIF1a).
Hypoxia and Cancer: Application highlight
Hypoxia is a common trait in tumors, attributable to insufficient vascularization. Cell response to hypoxic stress is primarily mediated through the transcription factor hypoxia inducible factor 1 (HIF-1). Low oxygen concentration induces HIF to regulate genes involved in restoring cellular homeostasis such as erythropoietin, glycolytic enzymes, and vascular endothelial growth factor (VEGF). Understanding the regulation and mechanisms of hypoxia-induced gene expression will provide critical insights into cancer pathogenesis.
Hypoxic conditions in tumors also cause resistance to radiotherapy and chemotherapy, leading to the recent pharmaceutical interest in anti-hypoxia drug development. Although HIF-1 inhibition in hypoxic tumor cells may confer therapeutic benefits, evaluating the full HIF-1 pathway is critical for efficient screening of small molecules and minimizing off-target effects.
The Power of the LightSwitch Luciferase Assay System
• Quantitative: Novel RenSP luciferase fusion technology allows you to measure promoter activity in response to Hypoxia pathway activation with industry-leading sensitivity and dynamic range
• Simple, quick, complete solution: Perform your experiments today through simple transfection of our luciferase reporter constructs using LightSwitch optimized reagents. No cloning, DNA preparation, or reagent optimization is needed. No need for co-tranfection of normalizing controls.
• Comprehensive and verified: Choose from SwitchGear’s complete set of sequence verified and transfection ready CREB targets.
• Functionally insightful: Learn about the actual effects of transcription factor binding to verify computational predictions and supplement microarray or sequencing data.
• Cost-effective: Profile promoter activation efficiently across the full Hypoxia pathway under a multitude of conditions in parallel.
Profiling Plate with 5 ug per well
HIF1a Pathway Validated Biomarker Set
LightSwitch GoClone Promoter Reporter Constructs & Controls
LightSwitch Luciferase Assay Reagents
Publication: Identification of Compounds which induce HIF1a
Webinar: Creating Reporter Panels, A Hypoxia Case Study
Background Info: Building Pathway Sets