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  • pCLuc-Basic 2 Vector

    Description

    pCLuc-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 Ostracod Cypridina noctiluca. Cypridina Luciferase (CLuc) is a 62 kDa protein with a native signal peptide at the N-terminus that allows it to be secreted from mammalian cells (1) so that CLuc activity can be detected in the culture medium. The pCLuc-Basic 2 Vector contains a multiple cloning site (MCS) upstream of the CLuc coding sequence. A neomycin resistance gene under the control of the SV40 promoter allows selection for stable integration of the plasmid into the mammalian cell genome using G418.

    Recommended sequencing primers for pCLuc-Basic 2:
    Upstream of MCS:
    pBasic Forward Sequencing Primer (23-mer) (not available from NEB)
    5´-GGGGTTCCGCGCACATTTCCCCG-3´(6020–6042)

    pBasic Reverse Primer (25-mer) (not available from NEB)
    5´-TCAGAAGCCATAGAGCCCACCGCAT-3´(1888–1864)

    CLuc 3´ End Forward Primer (23-mer) (not available from NEB)
    5´-GAGTTCAAGAAAGAATGCTACAT-3´(1671–1693)

    CLuc 5´ End Reverse Primer (24-mer) (not available from NEB)
    5´-GTAAGGACAGTCCTGGCAATGAAC-3´(143–120)
    Figure 1: Activity of Cypridina Luciferase in supernatants and lysates from a stable CLuc-expressing cell line. CLuc activity was measured from 20 µl of cell culture supernatant (500 µl total culture volume) and from 20 µl of cell lysate (100 µl total lysate volume).
    Figure 2: The high sensitivity of both the CLuc and GLuc assays allows detection of very small numbers of cells expressing each protein. 20 µl of culture supernatant from the indicated number of cells expressing each reporter were assayed.
    N0317

    Figure 3:  pCLuc-Basic 2 multiple cloning site (MCS). The Cypridina 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: 20–68
    • Start codon of CLuc: 75–77 Stop codon: 1734–1736
    • Signal peptide: 75–128
    • Synthetic poly-A site: 1745–1793
    • Neo promoter (SV 40): 2379–2714
    • Neomycin resistance gene: 2766–3560
    • Bacterial replication ori (pMB1): 4894–4306
    • Amp resistance: 5925–5065

    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 CLuc activity is found in the cell culture medium, with the remaining 5-10% detectable in cell lysates (Figure 1). This allows flexibility when assaying CLuc along with other co-transfected reporters.
    • The activity of CLuc is high and the CLuc assay is sensitive enough to detect very small amounts of CLuc enzyme activity (Figure 2).
    • CLuc does not use the same substrate as other marine luciferases (e.g. Renilla and Gaussia). Therefore, it is possible to assay both CLuc and GLuc independently in cell culture medium from cells expressing both reporters.
    • The pCLuc-Basic 2 Vector can be transfected into cells using any standard transfection protocol and stable cell lines can be established using Neomycin selection.

    Applications

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

    Properties and Usage

    Storage Temperature

    -20°C

    References

    1. Nakajima, Y. et al. (2004). Biosci. Biotechnol. Biochem. 63, 565-570.
    2. Wu, C., Suzuki-Ogoh, C. and Ohmiya, Y. (2007). BioTechniques. 42, 290-292.
    3. Otsuji, et at (2004). Anal. Biochemistry. 329, 230-237.

    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.
    1. Where can I find the sequence of this plasmid?
    2. Can I make a stable cell line with pCLuc-Basic 2?
    3. Can I transfect this plasmid into mammalian cells?
    4. How do I assay for CLuc expression?
    5. Can I use assay kits designed for other reporters (Gaussia, Renilla & Firefly luciferases) to assay CLuc activity?
    6. Is there another secreted reporter that can be used with CLuc?

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