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PAX6 Reporter Lentivirus
$595.00
PAX6 Reporter Lentivirus: A high quality lentiviral reporter system designed for robust detection of PAX6 transcriptional activity in mammalian cells. The reporter is specifically engineered to detect the synergistic activity of PAX6 with SOX2/3 by using tandem repeats of the Pax6 response element located within the LE9 enhancer. This enhancer is head surface ectoderm-specific and conserved across vertebrates, making it an effective tool for observing Pax activity, particularly in conjunction with Sox2/Sox3. The Pax6 gene is pivotal in the development of the eye and other sensory organs, as well as in certain neural and epidermal tissues, underscoring its essential role in development and differentiation processes. The reporter lentivirus is purified by PEG precipitation and sucrose gradient centrifugation and is ideal for difficult-to-transfect cells including primary and/or thawed cells.
Have questions about this product? Need a stable cell line? Send us a form and we’ll reply the same day: Contact Us
Ultra-high concentration virus can be provided upon request in your choice of medium and volume for in vivo injection. We can provide custom reporter genes (such as SEAP, YFP, secreted Gaussia Luc, Renilla Luc, etc.) and selection markers (hygromycin, bleomycin, etc.) upon request.
Available Options:
- Specifications
- Additional Information
- Vector Diagram
- Selecting Proper Controls
- MSDS
- Frequently Asked Questions
Specifications
Details
| LTV-0111 | |
| PAX6 signaling pathway | |
| PAX6 response elements | |
| Monitor PAX6 activity in mammalian cells, screen for activators or inhibitors of PAX6 mediated transcriptional activity. The fluorescent reporter enables convenient readout using flow cytometry, fluorescence microscopy, etc. | |
| Human/mouse |
Product Data
Figure 1. GFP Reporter activation of PAX6-TAG-Puro by PAX6 HEK293FT cells were co-transfected with plasmids for PAX6-TAG-Puro along with empty vector (pcDNA) or cDNA encoding human PAX6 for 32 h before acquiring fluorescent microscopy images. PAX6 expression vector contains RFP downstream of PAX6, separated by P2A. This product is supplied as pre-packaged lentiviral particles with your choice of reporter and selection marker. Featured cDNA expression vectors are also available upon request. For more information, please email info@lipexogen.com.
Have questions about this product? Send us a form and we’ll reply the same day: Contact Us
Background
The head surface ectoderm-specific enhancer, LE9, is an element found within the regulatory regions of the Pax6 gene, which plays a crucial role in the development of the eye, specifically in the lens and cornea. This enhancer is a sequence that is highly conserved across vertebrates and has been shown to be a highly effective enhancer in reporter analyses. The Pax6 protein interacts directly with the distal half of the LE9 sequence in a manner dependent on the paired domain of Pax6. Moreover, transcription factors Sox2 and Sox3, which contain HMG domains, can activate LE9 synergistically with Pax6, indicating a complex regulatory mechanism that involves direct and positive regulation of its own gene expression by Pax6. This interaction suggests a model where the transcriptional activation by Pax6 protein is modified by its interaction with Sox2 and Sox3 proteins (The BioGRID).
Additionally, the role of the ectoderm enhancer (EE) in the Pax6 gene extends beyond the regulation of Pax6 itself, impacting lens development significantly. Experiments have demonstrated that deleting this enhancer in mice leads to a range of defects in lens development, including thinner lens placodes, reduced cell proliferation, smaller lens vesicles, and delays in fiber cell differentiation. Despite these defects, lens development still occurs, albeit abnormally, suggesting the presence of other regulatory elements that contribute to Pax6 expression and lens development. These findings underscore the importance of the EE in normal lens formation and highlight a complex regulatory network involving multiple enhancers and transcription factors that drive the expression of Pax6 in distinct subdomains within the lens placode (The Biologists Journals).
The detailed interactions between Pax6, Sox2, Sox3, and the LE9 enhancer piece together a sophisticated mechanism of gene regulation that underscores the intricate genetic controls underlying eye development, particularly the formation of the lens and cornea. This area of research not only provides insights into developmental biology but also has implications for understanding and potentially treating developmental eye disorders.
Recommended Controls
Reporter Negative Control Lentivirus – Ready-to-transduce lentiviral particles expressing minimal TATA box-driven reporter. The construct is the same as the TF Reporters except that it lacks the transcriptional response elements which drive signal pathway/TF-specific reporter expression. The reporter negative control lentiviral particles allow to establish a baseline for background reporter activity and determine specificity of any treatments to activate the reporter.
Reporter Positive Control Lentivirus – Ready-to-transduce lentiviral particles with constitutive expression of the reporter. The reporter positive control lentivirus is useful for transduction optimization studies, especially if the cells are very sensitive or difficult-to-transduce.
Renilla Luciferase Internal Control Lentivirus – Ready-to-transduce lentiviral particles expressing minimal TATA box-driven Renilla luciferase (RLuc).
Custom Orders
If you require a modification to one of our products, such as a change in reporter or other vector component, please contact us. Examples of customization options are shown in the table below. Feel free to request something not in the table.
Additional Custom Service Options
- Send us your cells and we can establish a stable reporter cell line for you using this product. Learn more.
- ORF cDNA plasmids featured in the product figures are available upon request.
- Ultra-high concentration virus can be provided upon request in your choice of medium and volume (i.e. for in vivo applications).
Additional Information
Additional Information
- High Sensitivity – LipExoGen TF Reporter lentiviral particles are made using a novel vector platform based on the third generation system. Transcriptional response elements are arranged as DNA tandem repeats upstream of the minimal TATA promoter-driven reporter, and downstream of an optimized minimal enhancer (pc) of the human CMV promoter. When the signal pathway/TF is activated, the mini enhancer synergizes with TF binding to the response elements (up to 8 repeats in some products, depending on strength of reporter activation) to amplify expression of the fluorescent (GFP/RFP) or luciferase (Luc) reporter, with minimal enhancement of background. As a result, the reporter system provides a highly sensitive readout for signaling pathway or specific transcription factor activation in human and mouse cells.
- Easily Establish Stable Reporter Cell Lines – The reporter lentiviral particles are ultra-purified and concentrated to high-titer by PEG purification and sucrose gradient centrifugation to allow for efficient transduction of difficult-to-transfect cells, including primary and/or freeze-thawed cells. Stable cell lines are easily generated through puromycin or blasticidin selection.
- Discovery Made Easy – Signal pathway or specific transcription factor activity can be detected by fluorescence, making LipExoGen TF Reporter lentiviral particles more practical than traditional luciferase reporters and/or biochemical assays. Pathway/TF activation can be readout directly by fluorescence microscopy in living cell cultures, thus paving the way for unexpected discoveries.
- Readout On Flow – Fluorescent reporter activation can also be readout by flow cytometry, providing more versatility in data acquisition for labs with different instruments.
- Best Value – LipExoGen lentiviral particle products are made using optimized lentiviral vectors developed in-house, which allows us to provide the highest quality products while retaining competitive prices. These high-titer lentiviral particles feature a highly sensitive fluorescent reporter system which has been validated to read out the activity of the indicated transcription factor or signaling pathway.
- Same Cost For Custom Lentivirus – You can easily request any combination of reporter (GFP/RFP/Luc) and selection marker (puromycin/blasticidin) for this product, without additional cost, by contacting us. To view our complete list of vectors, click here.
Vector Diagram
Vector Diagram
Size: 6.5-8.0 kb
The reporter gene expression is driven by transcriptional response elements (arranged in tandem repeats) coupled to a minimal TATA promoter. The signal is boosted by the upstream enhancer element. Expression of the selection marker is driven separately by EF1a promoter.
Selecting Proper Controls
Selecting Proper Controls
Introduction
It is important to know if and when to use the proper controls for this product. The reporter lentivirus behavior will depend on the situation and therefore vary based on the conditions and cells it is being used in. We sell positive and negative control lentiviral particles which can be helpful in optimizing the conditions necessary to activate or inhibit the reporter activity, normalize luminescent signal (i.e. Renilla internal control), and troubleshoot the needed transduction conditions. Not everyone will need to use these controls, particularly if they are experienced with handling lentiviral reporters or perhaps if they have already purchased our product and determined ideal conditions for it already. However, we recommend using the proper controls always to ensure that the user can properly assess what they are seeing in their cells after transducing with our lentivirus. It should be noted that in some cases, an agonist (for example, TNF-alpha or LPS) may be needed to activate the reporter, and not all commercially-available agonists will work. We have had instances where TNF-alpha from one company cannot activate one of our reporters, whereas seemingly the same product from another company can. In these instances, we will do our best to inform of this on the website, however it is ultimately the responsibility of the buyer to figure these things out. If you observe something like this, we encourage you to contact us so that we can make the information available to others on our website.
The bottom line: When in doubt, use controls!
There are several different kinds of controls that you should consider when purchasing a reporter lentivirus from us. You have to decide which ones you will need, since it may depend on your particular application. Below is a general description which should help you in determining your individual needs. If you have questions, you can just contact us.
I. The Actual TF Reporter
Cat. No.: Various
Purpose: Experimental. This is the actual reporter that you will use to test the transcriptional activity of the indicated response elements in your experimental conditions. Example: test if a condition results in activation or inhibition of NFκB transcriptional activity.
Explanation of components:
Selection: Refers to the drug selection marker gene that is used to allow you to select the transduced cells and make a stable cell line. Currently you get 2 options, either puro or BSD.
Enhancer: This refers to an optimized minimal enhancer from the human CMV promoter. It is present in all of our TF Reporter constructs and functions by amplifying the reporter transcription driven by specific TF binding to response elements and the minimal TATA promoter.
TF Binding: Refers to the specific transcriptional response elements that are present to allow pathway or TF specific activation of the reporter. They are generally arranged as tandem repeats and the number of repeats varies depending on the product.
TATA: Minimal TATA promoter. Alone, it is insufficient to drive strong transcription of the reporter. It works together with the TF-bound response elements to drive the reporter expression when the specific signal pathway or TF is transcriptionally active.
Reporter: The fluorescent or luminescent reporter that is used to readout the pathway or TF activity in the transduced cells. Currently you can choose GFP, RFP, or Firefly Luciferase (FLuc).
II. Controls
Purpose: Establish a frame of reference for “positive reporter signal” that is independent of the pathway or TF-specific transcriptional response elements. The construct is organized the same as the actual TF reporters, except that a synthetic “constitutively active” TRE sequence is used to drive reporter activation in the cells. The reporter signal in the resultant cells can be used as a positive control to gauge whether the transduction conditions need to be optimized or if your particular test conditions are insufficient to activate the actual TF reporter.
Example 1: You are trying a specific ligand to induce the activity of your TF reporter, and not seeing much reporter activation. The positive control can help you determine if this is an issue with your transduction or if the ligand you selected is unable to activate the reporter. You may need to troubleshoot the ligand, add another ligand, co-transfect the cells with another gene, or something else.
Example 2: You are testing the inhibitory effect of your compound on a specific signaling pathway and see that it produces good inhibition. You can use the reporter positive control cells to confirm the effect of the compound is specific to that signaling pathway, not an off-target effect such as broad inhibition of transcription or translation.
How to use: Transduce a separate batch of cells (not the same ones that will receive the actual TF reporter). The reporter positive control should have the same selection marker and reporter as the actual TF reporter you are using to assay the desired transcriptional activity. This ensures you can control for any impact of the drug selection. Treat the transduced cells the same way as the ones that were transduced with the TF reporter. Use the reporter signal you observe to get an idea what reporter activation should look like in your cells.
Purpose: Establish a baseline for any background reporter activity.
How to use: Transduce a separate batch of cells (not the same ones that will receive the actual TF reporter). The reporter negative control should have the same selection marker and reporter as the actual TF reporter you are using to assay the desired transcriptional activity. This ensures you can control for any impact of the drug selection. Treat the transduced cells the same way as the ones that were transduced with the TF reporter. Any reporter activation or inhibition you observe represents the basal level (independent of transcriptional response elements).
RLuc Internal Control (Constitutive)
Purpose: Establish a frame of reference for “positive reporter signal” that is independent of the pathway or TF-specific transcriptional response elements. The advantage of the RLuc internal control is that it can be used in the same cells that receive the actual TF reporter construct driving Firefly luciferase. Thus, it can be in dual luciferase assays. The construct is organized the same as the actual TF reporters, except that a synthetic “constitutively active” TRE sequence is used to drive reporter activation in the cells. The reporter signal in the resultant cells can be used as a positive control to identify well-to-well variations since the Renilla luciferase can be detected separately from Firefly luciferase in the same batch of cells.
Example 1: You are trying a specific ligand to induce the activity of your TF reporter, and not seeing much Firefly luciferase reporter activation. If Renilla luc is also not being detected, it could indicate an issue with your cells, transduction/selection conditions, the instrument, or specific reagents being used. If you can detect RLuc but not FLuc, then it likely means the effect is pathway or TF-specific based on the TF reporter you are using. Note: to rule out reagent- or equipment-specific issues related to FLuc but not RLuc, you would need to transduce a separate batch of cells with Reporter Positive Control (LTV-0072) with FLuc as the reporter. The positive control can help you determine if this is an issue with your transduction or if the ligand you selected is unable to activate the reporter. You may need to troubleshoot the ligand, add another ligand, co-transfect the cells with another gene, or something else.
Example 2: You are testing the inhibitory effect of your compound on a specific signaling pathway and see that it produces good inhibition. You can use the constitutive RLuc internal control to verify that the effect of the compound is specific to that signaling pathway, not an off-target effect such as broad inhibition of transcription or translation.
How to use: Transduce the same batch of cells that will receive or already received the actual TF reporter. The RLuc Internal Control (Constitutive) should NOT have the same selection marker as the actual TF reporter you are using to assay the desired transcriptional activity. For example, if the actual TF reporter is using puro for selection, then the RLuc internal control should use blasticidin (BSD) and vice versa. This ensures you can select the same cells twice, once for the actual TF reporter, and again for the internal control. We strongly recommend that this be performed in two separate transductions. You should transduce the cells with one of the lentiviruses first, select the transduced cells, and let them grow up and completely recover before proceeding to the next round of transduction and selection with the second lentivirus. Details can be found in our transduction protocol which comes with each product and is also available under a different tab on this page.
RLuc Internal Control (Minimal)
Purpose: Establish a baseline for any background reporter activity in the same cells that contain the actual TF reporter construct. The advantage of the RLuc Internal Control is that it can be used in the same cells that receive the actual TF reporter construct driving Firefly luciferase. Thus, it can be used in dual luciferase assays. The construct is organized the same as the actual TF reporters, except that there are no transcriptional response elements to drive the expression of RLuc. Thus, any RLuc signal that is produced under your experimental conditions represents the basal activity of the construct independent of the transcriptional response elements (minimal TATA and upstream enhancer). This is useful in experiments where you are trying to study the reporter activation and need know the background level in dual-luciferase assays.
How to use: Transduce the same batch of cells that will receive or already received the actual TF reporter. The RLuc Internal Control (Minimal) should NOT have the same selection marker as the actual TF reporter you are using to assay the desired transcriptional activity. For example, if the actual TF reporter is using puro for selection, then the RLuc internal control should use blasticidin (BSD), and vice versa. This ensures you can select the same cells twice, once for the actual TF reporter, and again for the internal control. We strongly recommend that this be performed in two separate transductions. You should transduce the cells with one of the lentiviruses first, select the transduced cells, and let them grow up and completely recover before proceeding to the next round of transduction and selection with the second lentivirus. Details can be found in our transduction protocol which comes with each product and is also available under a different tab on this page.
Lentiviral Transduction Protocol
MSDS
Frequently Asked Questions
Frequently Asked Questions
Can I purchase the plasmid for this product?
TF reporter products such as this one are not available in plasmid format. They are only available as high-titer lentiviral particles. Other gene products on our site, such as ORF cDNA and shRNA products are available in either format.