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  • pGLuc-Basic 2 Vector

    Description

    pGLuc-Basic 2 is a cloning vector for expression in mammalian cells, containing a reporter gene but lacking promoter elements. The reporter gene is the secreted luciferase from the copepod Gaussia princeps. Gaussia Luciferase (GLuc) is a 19 kDa protein encoded by a "humanized" sequence, and it contains a native signal peptide at the N-terminus that allows it to be secreted from mammalian cells into the cell culture medium (1,2). The pGLuc-Basic 2 Vector contains a multiple cloning site (MCS) upstream of the GLuc coding sequence. A neomycin resistance gene under the control of an SV40 promoter allows selection for stable integration of the plasmid into the mammalian cell genome using G418.

    N8082
    Figure 1: Comparison of light output obtained from HEK293 cells transfected with either the promoterless pGLuc-Basic 2 Vector or the pCMV-GLuc 2 Control Plasmid (NEB #N8081). Supernatants were harvested at 24 hour post-transfection and assayed for the GLuc activity using the BioLux GLuc Assay System.

    N8082
    N8082
    Figure 2: pGLuc-Basic multiple cloning site (MCS). The Gaussia Luciferase sequence is shown with a blue background. Only unique restriction sites are shown.

    DNASU is a central repository for plasmid clones and collections that may also be helpful.

    Highlights

    • Polylinker MCS: 12–68
    • GLuc coding: 76–633
    • Start codon: 76–78
    • Stop codon: 631–633
    • Signal peptide: 76–126
    • Synthetic poly-A site: 642–690
    • Neo promoter (SV40): 1276–1611
    • Neomycin resistance gene: 1663–2457
    • Bacterial replication ori (pMB1): 3791–3203
    • Amp resistance: 4822–3962
    • All pGLuc 2 vectors and plasmids have improved polyadenylation-transcription termination of the luciferase transcript. The polyadenylation signal is a synthetic polyadenylation sequence based on the β-globin gene (5)

    Product Source

    Isolated from an E. coli strain NEB10b by standard DNA purification procedure.

    Advantages and Features

    Features

    • Multiple samples can be obtained from the same transfected cells (i.e., before and after experimental treatments or at multiple time points).
    • 90–95% of GLuc activity is found in the cell culture medium, with the remaining 5–10% detectable in cell lysates. This allows flexibility when assaying GLuc along with other co-transfected reporters.
    • The activity of GLuc is high and the GLuc assay is sensitive enough to detect very small amounts of GLuc enzyme activity.
    • GLuc is very stable in the cell culture medium so the GLuc activity detected reflects the amount of GLuc secreted by the transfected cells over a period of several days. GLuc can also be stored at 4°C for several days without any loss in activity.
    • GLuc does not use the same substrate as Cypridina Luciferase. Therefore, it is possible to assay both GLuc and CLuc independently in cell culture medium from cells expressing both reporters (3,4).
    • The pGLuc-Basic 2 Vector can be transfected into cells using any standard transfection protocol and stable cell lines can be established using Neomycin selection.
    Recommended Sequencing Primers for pGLuc-Basic 2 Vector (not available from NEB)
    Upstream of MCS: (23-mer)
    5´-GGGGTTCCGCGCACATTTCCCCG-3´(4917–4939)

    pBasic Reverse Primer (25-mer)
    5´-TCAGAAGCCATAGAGCCCACCGCAT-3´(785–761)

    GLuc 3´ End Forward Primer (20-mer)
    5´-GCCAGCAAGATCCAGGGCCA-3´(580–599)

    GLuc 5´ End Reverse Primer (24-mer)
    5´-TCAGGGCAAACAGAACTTTGACTC-3´(103–80)

    Applications

    • The pGLuc-Basic 2 Vector can be used to test promoters by cloning promoter element of interest into the MCS upstream of the GLuc reporter gene. For constitutive expression of GLuc, vectors containing promoters are available (See Companion Products Sold Separately).
    • GLuc can be used as a stand alone reporter or in conjunction with other compatible reporters such as Cypridina Luciferase (CLuc) (3). GLuc and CLuc are ideally suited for co-expression as both are secreted and highly active enzymes providing ease of use and sensitivity (3,4).

    Properties and Usage

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    1 mM EDTA
    pH 7.5 @ 25°C

    References

    1. Verhaegen, M. and Christopoulos, T.K. (2002). Anal. Chem.. 74, 4378-4385.
    2. Tannous, B.A. et al. (2005). Mol. Ther.. 11, 435-443.
    3. Otsuji, et al. (2004). Anal. Biochemistry. 329, 230-237.
    4. Wu, et al. (2007). Biotechniques. 42, 290-292.
    5. Levitt, et al. (1989). Genes Dev.. 3, 1019-1025.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Where can I find the sequence of this plasmid?
    2. Can I generate a stable cell line with pGLuc-Basic 2 Vector?
    3. Can I transfect this plasmid into mammalian cells?
    4. How do I assay for GLuc expression?
    5. Is there another secreted reporter that can be used with GLuc?

    Selection Tools

    Interactive Tools

    Application Notes