载体描述： The Mammalian Lumio Gateway Vector Kits contain Gateway-adapted destination vectors designed for use with the Lumio Technology. The pcDNA6.2/Lumio-DEST vectors supplied with each kit facilitate in vivo fluorescence labeling and detection of recombinant proteins when used with a Lumio In-Cell Labeling Kit. 载体特征： The pcDNA6.2/cLumio-DEST and pcDNA6.2/nLumio-DEST vectors contain the following elements:
Human cytomegalovirus immediate-early (CMV) promoter/enhancer for high-level expression in a wide range of mammalian cells
Lumio tag for C-terminal (pcDNA6.2/cLumio-DEST) or N-terminal (pcDNA6.2/nLumio-DEST) fusion to the gene of interest for fluorescence detection
Two recombination sites, attR1 and attR2, downstream of the CMV promoter for recombinational cloning of the gene of interest from an entry clone
Chloramphenicol resistance gene located between the two attR sites for counterselection
The ccdB gene located between the two attR sites for negative selection
The Herpes Simplex Virus thymidine kinase polyadenylation signal for proper termination and processing of the recombinant transcript
f1 intergenic region for production of single-strand DNA in F plasmidcontaining E. coli
SV40 early promoter and origin for expression of the Blasticidin resistance gene and stable propagation of the plasmid in mammalian hosts expressing the SV40 large T antigen
Blasticidin resistance gene for selection of stable cell lines
The pUC origin for high copy replication and maintenance of the plasmid in E. coli
The ampicillin resistance gene for selection in E. coli Gateway 技术简介： The Gateway Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (Landy, 1989) to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems. To express your gene of interest in mammalian cells using Gateway Technology, simply:
1. Clone your gene of interest into a Gateway entry vector to create an entry clone.
2. Generate an expression clone by performing an LR recombination reaction between the entry clone and a Gateway destination vector (e.g. pcDNA6.2/cLumio-DEST or pcDNA6.2/nLumio-DEST).
3. Transfect your expression clone into the cell line of choice for transient or stable expression of your gene of interest. Lumio 技术的优势： The Lumio System is based on the FlAsH (Fluorescein Arsenical Hairpin) technology which uses biarsenical labeling reagents to bind and detect proteins containing a tetracysteine motif (i.e. Lumio tag) (Griffin et al., 1998). Using the Lumio Technology and the Lumio In-Cell Labeling Kits for fluorescence labeling of recombinant proteins provides the following advantages:
Small size of the Lumio tag (6 amino acids, 585 Da) is less likely to interfere with the structure or biological activity of the protein of interest
Lumio Labeling Reagents are membrane-permeable and readily cross the cell membrane, allowing labeling and detection of recombinant proteins in live mammalian cells
Lumio Labeling Reagents bind the Lumio tag with high specificity and high affinity (nanomolar or lower dissociation constant), allowing targeted labeling of the protein of interest
Lumio Labeling Reagents become strongly fluorescent only upon binding the Lumio tag, allowing specific detection of Lumio-tagged proteins Lumio 系统的组成： The Lumio System consists of two major components:
The tetracysteine Lumio tag (Cys-Cys-Pro-Gly-Cys-Cys) in the pcDNA6.2/Lumio-DEST vector. When fused to a gene of interest, the Lumio tag allows the expressed fusion protein to be specifically recognized by a biarsenical labeling reagent. For more information on the tetracysteine motif, see below.
A biarsenical labeling reagent, Lumio Green or Lumio Red, which becomes fluorescent upon binding to recombinant proteins containing the Lumio tag.
The Lumio Green and Lumio Red Labeling Reagents are supplied precomplexed to the dithiol EDT (1,2-ethanedithiol) which stabilizes and solubilizes the biarsenic reagents.
Tetracysteine Motif
The Lumio Reagents bind a tetracysteine motif consisting of Cys-Cys-Xaa-XaaCys-Cys where Cys equals cysteine and Xaa equals any amino acid other than cysteine. This motif is rarely seen in naturally occurring proteins allowing specific fluorescence labeling of recombinant proteins fused to the Lumio tag. In the Lumio System, the optimized Cys-Cys-Pro-Gly-Cys-Cys tetracysteine motif is used as this motif has been shown to have a higher affinity for and more rapid binding to biarsenic compounds as well as enhanced stability compared to other characterized motifs (Adams et al., 2002).
Lumio Green Detection Kit
For sensitive and specific in-gel detection of Lumio-tagged fusion proteins, we recommend the Lumio Green Detection Kit available from Invitrogen (Catalog no. LC6090). The Lumio Green Detection Kit enables immediate visualization of Lumio-tagged proteins in polyacrylamide gels using a UV transilluminator or a visible light laser-based scanner and without the need for staining or western blotting. In addition, the BenchMark Fluorescent Protein Standard (Catalog no.LC5928) allows you to easily visualize molecular weight ranges of proteins labeled with Lumio Green Detection Reagent. 实验要点： Generating an Entry Clone
Introduction To recombine your gene of interest into pcDNA6.2/cLumio-DEST or pcDNA6.2/nLumio-DEST, you will need an entry clone containing the gene of interest. Many entry vectors including pENTR/D-TOPO (Catalog no. K2400-20) are available from Invitrogen to facilitate generation of entry clones.
Tag-On-Demand System
The pcDNA6.2/cLumio-DEST vector is compatible with the Tag-OnDemand System which allows expression of both native and C-terminallytagged recombinant protein from the same expression construct.
The System is based on stop suppression technology originally developed by RajBhandary and colleagues (Capone et al., 1985) and consists of a recombinant adenovirus expressing a tRNAser suppressor. When an expression vector encoding a gene of interest with the TAG (amber stop) codon is transfected into mammalian cells, the stop codon will be translated as serine, allowing translation to continue and resulting in production of a C-terminally-tagged fusion protein.
If you wish to express a human or mouse gene of interest, we recommend using an Ultimate Human ORF (hORF) or Ultimate Mouse ORF (mORF) Clone available from Invitrogen. Each Ultimate ORF Clone is a fully sequenced clone provided in a Gateway entry vector that is ready-to-use in an LR recombination reaction with pcDNA6.2/cLumio-DEST. In addition, each clone contains a TAG stop codon, making it fully compatible for use in the Tag-On-Demand System.
Points to Consider for pcDNA6.2/nLumio-DEST
pcDNA6.2/nLumio-DEST allows expression of recombinant proteins with an N-terminal peptide containing the Lumio and V5 epitope tags and contains an ATG initiation codon within the context of a Kozak consensus sequence. To include the Lumio and V5 epitope tags, your insert in the entry clone should:
not contain a Kozak initiation sequence
be in frame with the Lumio and V5 epitope tags after recombination
contain a stop codon
