Properties


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Overview
  • Xactagen vector designs, reporter genes, and assay reagents all combine to provide higher signal to noise (Z-factors) and ease of use compared to competitive products – both in vitro and in vivo. Included is membrane-anchored Gaussia luciferase, displayed on the outside of the cell and self-stabilized for unsurpassed glow activity. The result is the ability to monitor gene expression of not only moderately and highly expressed genes, but also weakly expressed genes below the threshold for detection by other reporter systems (e.g. sensitivity for up to 50% more gene promoters).

  • Data comparing Xactagen reporters, vectors and assay reagents to competitive products follows our description of unique attributes for Xactagen reporter genes, exogenous reporter vectors, and endogenous reporter cells (presented below). Also, please see Vector Designs and Ease of Use for more information on these topics.


Reporter Genes:
    Secreted Gaussia luciferase

  • Secreted Gaussia luciferase is an ATP independent and highly stable enzyme secreted into the medium of cultured cells or into the blood stream of animal models. It has the highest activity of any characterized luciferase, with up to 1,000-fold more activity than firefly or Renilla luciferase (Tannous et. al., 2005). The enzyme out performs other luciferases both in vitro and in vivo, including use with whole-animal imaging technologies. Both flash and glow assays are available with Xactagen assay reagents. For more information see supporting data below and Secreted Gaussia Luciferase.


  • Membrane-anchored Gaussia luciferase

  • Membrane-anchored Gaussia luciferase is anchored to the cell membrane and displayed on the outside of the cell. It is self-stabilized for glow activity (e.g. no chemical stabilizers are required), and its glow activity is >10-fold higher than glow activity of secreted Gaussia luciferase – making it the highest glow activity of any characterized luciferase. The enzyme also has a reduced half-life, so that effects of activators and inhibitors can be measured without accumulated reporter activity that can sometimes mask their effects. For more information see supporting data below and Membrane-Anchored Gaussia Luciferase.


  • Ptilosarcus green fluorescent protein (Pt-GFP)

  • This fluorescent protein is up to three times more active than enhanced green fluorescent protein (EGFP). Combined with Xactagen vector designs, Pt-GFP provides exceptional signal to noise.


Endogenous or Exogenous Reporter Cells:
    Endogenous Reporter Cells

  • Endogenous reporter cells are the most reliable means of indexing gene expression using reporter genes. They account for native promoters, enhancers and silencers, some of which can reside tens of thousands of base pairs from the transcription start site. They also account for local chromosomal influences on gene expression, such as DNA methylation. Although desirable, few endogenous reporter cells are commercially available from other vendors: they require very sensitive reporter genes and vector systems and they historically have been difficult and time-consuming to produce.

  • Xactagen vector designs and reporter genes now provide the high signal to noise (Z-factors) required for monitoring not only moderately and highly expressed genes, but also weakly expressed genes. Included is the option to use membrane-anchored Gaussia luciferase with its exceptional flash and self-stabilized glow activity. And because Xactagen’s endogenous reporter constructs conserve native mRNA sequences including coding exons and 5’ and 3’ untranslated regions (UTRs), Xactagen reporters are also under control of micro-RNAs in cells. Furthermore, Xactagen technology advances enable production of endogenous reporter cells for most any gene or set of genes (e.g. genes regulated in signaling pathway, drug-responsive genes, siRNA responsive genes, etc.).
  • Exogenouse Reporter Vectors/Cells

  • Xactagen offers ectopic reporter vectors and reporter cells utilizing Xactagen vector designs and reporter genes to provide higher sensitivities and ease of use compared to competitive products. As a result, Xactagen vectors now enable analysis of weak promoters that other vectors can not, and provide more accurate expression data for moderately and highly expressing promoters/genes – both in vitro and in vivo.



Performance Data:
    Publication List

  • We have compiled a Gaussia Luciferase Publication List. Data comparing Gaussia luciferase to firefly and Renilla luciferase can be found as well as research applications. Some figure excerpts from Tannous et. al., 2005 are provided in the following links: in vitro comparison, in vivo comparison


  • Performance of Xactagen Vector Designs and Assay Reagents

  • HeLa cells were transfected with Xactagen’s (XGEN) pCMV-sGL.1 to transiently express secreted Gaussia luciferase or with a comparable vector from a competitive vendor (Comp. Vend.) (1ug per well, 6-well culture plates, lipofection). Medium on cells was replaced with fresh growth medium after 6 hours of transfection and then again after 36 hours. Medium was attained for flash Gaussia luciferase assays 48 hours after initiating transfection (5uL of medium per assay) and assayed using either Xactagen assay reagents or those from a competitive vendor. Assays were performed in quadruplicate.

The data demonstrate that Xactagen vectors and assay reagents produce ~14-fold more Gaussia luciferase enzyme activity as compared to vectors and assay reagents form a competitive vendor.

    ~2.5-fold was attributed to assay reagents

    ~5.9-fold was attributed to vector design


Figure 1: Performance of Xactagen vectors and assay reagents compared to those from a competitive vendor.

    More Glow Activity with Xactagen Reporters

  • HeLa cells were transfected with Xactagen vectors (pCMV-mGL.1 to express membrane-anchored Gaussia luciferase or pCMV-sGL.1 to express secreted Gaussia luciferase) or a vector from a competitive vendor (pCMV-GLuc to express secreted Gaussia luciferase). Transfections used 1ug per well, in 6-well culture plates, lipofection). For vectors expressing secreted Gaussia luciferase, medium on cells was replaced with fresh growth medium 36 hours after intitiating transfection and 5uL attained for kinetic assays 48 hours after initiating transfection. For vectors expressing membrane-anchored Gaussia luciferase, transfected cells were seeded into 384-well cell culture treated assay plates. Assays were performed 48 hours after initiating transfection. When using Xactagen vectors, Xactagen assay reagents were utilized. When using the competitive vendor’s vector, the competitive vendor’s assay reagents with or without chemical stabilizers were utilized. Assays were performed in quadruplicate with a Molecular Devices (Sunnyvale, CA) SpectraMaxL luminescence plate reader (Figure 2).


Figure 2: Glow properties of Xactagen vectors and reagents compared to those of a competitive vendor.

  • Xactagen’s vectors and assay reagents resulted in higher enzyme activity compared to the competitive vendor’s vectors and reagents.
  • Xactagen’s membrane-anchored Gaussia luciferase had >40-fold more enzyme activity than the competitive vendor’s products – whether or not a chemical stabilizer was used.
  • Xactagen’s membrane-anchored Gaussia luciferase is self-stabilized: it does not require addition of a chemical stabilizer.

    High Signal to Noise (Z-factors)

  • Transfections were performed for 8 hours using GenJetTM Reagent (Ver.II) from SignaGen Laboratories, Gaithersburg, MD following manufacturer’s protocols. Cell culture medium was replaced after 24 hours. Flash assays were performed with Xactagen’s GLum.1 assay kit at 48 hours in 96 well plates using 10uL of medium (secreted Gaussia luciferase: sGL.1) or ~8,000 cells (destabilized, membrane-anchored Gaussia luciferase: mGL.1). Assays were performed with Molecular Devices (Sunnyvale, CA) SpectraMaxL luminescence plate reader.

Figure 3: Signal to noise (Z-factors) for Xactagen's secreted or membrane-anchored Gaussia luciferase reporters using Xactagen's Glum.1 assay reagents.

  • The data demonstrates that very high signal to noise (Z-factors) are attained with Xactagen vectors and assay reagents - for both secreted and membrane-anchored Gaussia luciferase. Assays are accordingly amenable to bench top and automated screening. Although signal is stronger with secreted Gaussia luciferase, background from empty vector (MCS-vector) is lower with membrane-anchored Gaussia luciferase resulting in >6-fold higher signal to noise.

    Large Range of Expression for Endogenous Genes

  • Human colon cancer cells were transfected with Xactagen’s gene trap vector, pXGN101 containing membrane-anchored Gaussia luciferase reporter. Representative G418 resistant clones were expanded and evaluated for flash enzyme activity (6,000 cells per well: 384-well format). Presented activities are those after subtracting background luminescence (~120 rlu).


Figure 4: Expression levels of representative HCT116 human colon cancer reporter cell clones.

  • Expression levels spanned from 2-fold background to over 5,500-fold background (spanning ~100 RLUs over background to ~6.5 million RLUs over background) reflecting a large range of gene expression activities for endogenous genes.

  • Sensitivity of Xactagen vectors and reporter genes combined with low background luminescence provided by Xactagen assay reagents enable analysis of very weak promoters as well as very highly active promoters. We estimate that ~50% of transcription units in mammalian cells are driven by weak promoters that are below the threshold for detection by other reporters, vectors and assay reagents.