virapower_lentiviral_lumio_man

user guide For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. ViraPower™ II Lentiviral Lumio™ Gateway® Vectors Gateway® destination vectors for cloning and high- level expression using the ViraPower™ II Lentiviral Expression System, and site-specific fluorescence labeling and detection using Lumio™ Technology Catalog numbers K370-20 and K371-20 Revision date 29 March 2012 Publication Part number 25-0860 MAN0000529 ii iii Contents Kit Contents and Storage ...................................................................................................................................... v Introduction ................................................................................................................... 1 Product Overview .................................................................................................................................................. 1 Methods ......................................................................................................................... 4 Generating an Entry Clone .................................................................................................................................... 4 Creating Expression Clones .................................................................................................................................. 6 Performing the LR Recombination Reaction ...................................................................................................... 9 Transforming One Shot® Stbl3™ Competent E. coli .......................................................................................... 11 Guidelines for Expression ................................................................................................................................... 13 Detection and Analysis ........................................................................................................................................ 16 Appendix ...................................................................................................................... 18 Recipes ................................................................................................................................................................... 18 Blasticidin .............................................................................................................................................................. 19 Map of pLenti6.2/C-Lumio™/V5-DEST ............................................................................................................ 20 Map of pLenti6.2/N-Lumio™/V5-DEST ........................................................................................................... 21 Features of pLenti6.2/N- and C-Lumio™/V5-DEST Vectors ......................................................................... 22 Map of pLenti6.2/C-Lumio™/V5-GW/lacZ ..................................................................................................... 23 Map of pLenti6.2/N-Lumio™/V5-GW/lacZ..................................................................................................... 24 Accessory Products .............................................................................................................................................. 25 Technical Support ................................................................................................................................................. 26 Purchaser Notification ......................................................................................................................................... 27 References .............................................................................................................................................................. 29 iv v Kit Contents and Storage Types of Kits This manual is supplied with the following products. Product Catalog no. ViraPower™ II Lentiviral C-Lumio™ Gateway® Expression System K370-20 ViraPower™ II Lentiviral N-Lumio™ Gateway® Expression System K371-20 ViraPower™ II Lentiviral Lumio™ Gateway® Vectors The vectors provided with each catalog number are listed below. All vectors are provided in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. Store the vectors at –20°C. Catalog no. Vector Concentration Amount K370-20 pLenti6.2/C-Lumio™/V5-DEST pLenti6.2/ C-Lumio™/V5-GW/lacZ control 150 ng/µL 0.5 µg/µL 6 µg 10 µg K371-20 pLenti6.2/N-Lumio™/V5-DEST pLenti6.2/N-Lumio™/V5-GW/lacZ control 150 ng/µL 0.5 µg/µL 6 µg 10 µg Additional Kit Components In addition to the vectors listed above, the ViraPower™ II Lentiviral Lumio™ Gateway® Expression System kits include the following components. For a detailed description of the reagents supplied with the One Shot® Stbl3™ Chemically Competent E. coli, see page vi. Component Amount Storage Lumio™ Green In-Cell Labeling Kit 1 kit –20°C, protected from light One Shot® Stbl3™ Chemically Competent E. coli 20 rxns (1 kit) –80°C ViraPower™ Bsd Lentiviral Support Kit 20 rxns (1 kit) Blasticidin: –20°C ViraPower™ Packaging Mix: –20°C Lipofectamine® 2000: 4°C 293FT Cell Line 3 × 106 cells (1 kit) Liquid nitrogen Product Use For research use only. Not intended for any human or animal therapeutic or diagnostic use. Continued on next page vi Kit Contents and Storage, Continued One Shot® Stbl3™ Chemically Competent E. coli The following reagents are included with the One Shot® Stbl3™ Chemically Competent E. coli kit. Transformation efficiency is ≥ 1 × 108 cfu/µg plasmid DNA. Store at –80°C. Reagent Composition Amount S.O.C. Medium 2% Tryptone 0.5% Yeast Extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 20 mM glucose 6 mL Stbl3™ Cells – 21 × 50 µL pUC19 Control DNA 10 pg/µL in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8 50 µL Genotype of Stbl3™ Cells F– mcrB mrr hsdS20(rB–, mB–) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(StrR) xyl-5 λ– leu mtl-1 ViraPower™ Bsd Lentiviral Support Kit and 293FT Cell Line For a detailed description of the reagents provided in the ViraPower™ Bsd Lentiviral Support Kit and their use, refer to the ViraPower™ Lentiviral Expression System manual. For a detailed description of the 293FT Cell Line and instructions to culture and maintain the cells, refer to the 293FT Cell Line manual. Both manuals are included separately with this kit and also available for downloading from www.lifetechnologies.com/support or by contacting Technical Support (see page 26). Lumio™ Green In-Cell Labeling Kit ViraPower™ II Lentiviral Lumio™ Gateway® Expression System Kits are supplied with the Lumio™ Green In-Cell Labeling Kit. Refer to the Lumio™ In-Cell Labeling Kits manual for detailed information pertaining to this kit. 1 Introduction Product Overview Description of the System The pLenti6.2/N- and C-Lumio™/V5-DEST vectors are Gateway®-adapted destination vectors for use with Lumio™ Technology, and are designed to allow high-level expression of recombinant fusion proteins in dividing and non- dividing mammalian cells using our ViraPower™ II Lentiviral Expression System. The vectors supplied with each kit facilitate in vivo fluorescence labeling and detection of recombinant proteins using the Lumio™ Green In-Cell Labeling Kit. Features of the Vectors The pLenti6.2/N- and C-Lumio™/V5-DEST vectors contain the following elements: • Rous Sarcoma Virus (RSV) enhancer/promoter for Tat-independent production of viral mRNA in the producer cell line (Dull et al., 1998) • Modified HIV-1 5′ and 3′ Long Terminal Repeats (LTR) for viral packaging and reverse transcription of the viral mRNA (Dull et al., 1998; Luciw, 1996) Note: The U3 region of the 3′ LTR is deleted (∆U3) and facilitates self-inactivation of the 5′ LTR after transduction to enhance the biosafety of the vector (Dull et al., 1998) • HIV-1 psi (Ψ) packaging sequence for viral packaging (Luciw, 1996) • HIV Rev response element (RRE) for Rev-dependent nuclear export of unspliced viral mRNA (Kjems et al., 1991; Malim et al., 1989) • Lumio™ tag for C-terminal (pLenti6.2/C-Lumio™/V5-DEST) or N-terminal (pLenti6.2/N-Lumio™/V5-DEST) fusion to the gene of interest for fluorescence detection • Human cytomegalovirus immediate-early (CMV) promoter for constitutive expression of the gene of interest from a viral promoter (see page 2) • 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 (CmR) located between the two attR sites for counterselection • The ccdB gene located between the attR sites for negative selection • V5 epitope for detection of the recombinant protein of interest (Southern et al., 1991) using anti-V5 antibodies • PGK promoter driving expression of the Blasticidin resistance gene • Blasticidin (Izumi et al., 1991; Kimura et al., 1994; Takeuchi et al., 1958; Yamaguchi et al., 1965) resistance gene for selection of stable cell lines • Ampicillin resistance gene for selection in E. coli • pUC origin for high-copy replication of the plasmid in E. coli A control plasmid containing the lacZ gene is included with each vector for use as a positive expression control in the mammalian cell line of choice. For more information, see pages 23–24. Continued on next page 2 Product Overview, Continued CMV Promoter The pLenti6.2/N- and C-Lumio™/V5-DEST vectors contain the human CMV immediate early promoter to allow high-level, constitutive expression of the gene of interest in mammalian cells (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987). Although highly active in most mammalian cell lines, activity of the viral promoter can be down-regulated in some cell lines due to methylation (Curradi et al., 2002), histone deacetylation (Rietveld et al., 2002), or both. Gateway® Technology 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 the Gateway® Technology, simply: 1. Clone your gene of interest into a Gateway® entry vector of choice 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., pLenti6.2/N-Lumio™/V5-DEST or pLenti6.2/C-Lumio™/V5-DEST). 3. Use your expression clone in the ViraPower™ Lentiviral Expression System (see below). For detailed information about the Gateway® Technology, refer to the Gateway® Technology with Clonase® II manual which is available from www.lifetechnologies.com/support or by contacting Technical Support (see page 26). ViraPower™ II Lentiviral Expression System The ViraPower™ II Lentiviral Expression System facilitates highly efficient, in vitro or in vivo delivery of a target gene to dividing and non-dividing mammalian cells using a replication-incompetent lentivirus. Based on the lentikat™ system developed by Cell Genesys (Dull et al., 1998), the ViraPower™ II Lentiviral Expression System possesses features which enhance its biosafety while allowing high-level gene expression in a wider range of cell types than traditional retroviral systems. To express your gene of interest in mammalian cells using the ViraPower™ II Lentiviral Expression System, perform the following steps: 1. Create an expression clone in pLenti6.2/N- or C-Lumio™/V5-DEST. 2. Cotransfect your expression clone and the ViraPower™ Packaging Mix into the 293FT Cell Line to produce lentivirus. 3. Use your lentiviral stock to transduce the mammalian cell line of choice. 4. Assay for “transient” expression of the recombinant protein or generate a stable cell line using Blasticidin selection. For more information about the ViraPower™ II Lentiviral Expression System, the ViraPower™ Packaging Mix, and the biosafety features of the System, refer to the ViraPower™ Lentiviral Expression System manual. For more information about the 293FT Cell Line, refer to the 293FT Cell Line manual. Continued on next page 3 Product Overview, Continued Advantages of Lumio™ Technology 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 Components of the Lumio™ System The Lumio™ System consists of two major components: • The tetracysteine Lumio™ tag (Cys-Cys-Pro-Gly-Cys-Cys) in the pLenti6.2/N- or C-Lumio™/V5-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. • A biarsenical labeling reagent, Lumio™ Green, which becomes fluorescent upon binding to recombinant proteins containing the Lumio™ tag. Lumio™ Green Labeling Reagent is supplied pre-complexed to the dithiol EDT (1,2- ethanedithiol), which stabilizes and solubilizes the biarsenic reagent. Tetracysteine Motif The Lumio™ Reagents bind a tetracysteine motif consisting of Cys-Cys-Xaa-Xaa- Cys-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). Detecting In-Gel Lumio™ For sensitive and specific in-gel detection of Lumio™-tagged fusion proteins, we recommend the Lumio™ Green Detection Kit available (see page 25 for ordering). 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 (see page 25 for ordering) allows you to easily visualize molecular weight ranges of proteins labeled with Lumio™ Green Detection Reagent. For more information, visit www.lifetechnologies.com or contact Technical Support (page 26). 4 Methods Generating an Entry Clone Introduction To recombine your DNA sequence of interest into pLenti6.2/N-Lumio™/V5-DEST or pLenti6.2/C-Lumio™/V5-DEST, you will need to generate an entry clone containing the DNA sequence of interest. Many entry vectors are available for purchase to facilitate generation of entry clones (see table below for a representative list). For more information about these and other entry vectors, see www.lifetechnologies.com or contact Technical Support (see page 26). Once you have selected an entry vector, refer to the manual for the specific entry vector you are using for instructions to construct an entry clone. All entry vector manuals are available for downloading from www.lifetechnologies.com/support or by contacting Technical Support (see page 26). Entry Vector Catalog no. pENTR™/D-TOPO® K2400-20 pENTR™/SD/D-TOPO® K2420-20 pENTR™/TEV/D-TOPO® K2525-20 If you wish to express a human gene of interest in a pLenti6.2/N- or C- Lumio™/V5-DEST vector, you may want to use an Ultimate™ Human ORF (hORF) Clone available for purchase. The Ultimate™ hORF Clones are fully sequenced clones provided in a Gateway® entry vector that is ready to use in a recombination reaction with a pLenti-DEST vector. For more information about the Ultimate™ hORF Clones available, see www.lifetechnologies.com or contact Technical Support (see page 26). Kozak Consensus Sequence If you will be expressing your protein from pLenti6.2/C-Lumio™/V5-DEST, your insert in the entry clone should contain a Kozak translation initiation sequence with an ATG initiation codon for proper initiation of translation (Kozak, 1987; Kozak, 1990; Kozak, 1991). An example of a Kozak consensus sequence is provided below. The ATG initiation codon is shown underlined. (G/A)NNATGG Other sequences are possible, but the G or A at position –3 and the G at position +4 are the most critical for function (shown in bold). Continued on next page 5 Generating an Entry Clone, Continued Points to Consider for pLenti6.2/C- Lumio™/V5-DEST pLenti6.2/C-Lumio™/V5-DEST allows expression of recombinant proteins with a C-terminal peptide containing the V5 epitope and the Lumio™ tag; however, you may use this vector to express a native protein, if desired. Consider the following when generating your entry clone. If you wish to... Then your insert... include the V5 epitope and Lumio™ tag • should contain a Kozak initiation sequence (see page 4) • should not contain a stop codon • should be in frame with the V5 epitope and Lumio™ tag after recombination (see page 7 for a diagram) not include the V5 epitope and Lumio™ tag • should contain a Kozak initiation sequence (see page 4) • should contain a stop codon Points to Consider for pLenti6.2/N- Lumio™/V5-DEST pLenti6.2/N-Lumio™/V5-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 (see page 4). 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 (see page 8 for a diagram) • should contain a stop codon 6 Creating Expression Clones Introduction After you have generated an entry clone, you will perform the LR recombination reaction to transfer the gene of interest into pLenti6.2/N- or C-Lumio™/V5-DEST to create your expression clone. To ensure that you obtain the best possible results, we recommend that you read this section and the sections entitled Performing the LR Recombination Reaction (pages 9–12) and Transforming One Shot® Stbl3™ Competent E. coli (pages 11–12) before beginning. Experimental Outline To generate an expression clone: 1. Perform an LR recombination reaction using the attL-containing entry clone and the attR-containing pLenti6.2/N-Lumio™/V5-DEST or pLenti6.2/C-Lumio™/V5-DEST vector. Note: Both the entry clone and the destination vector should be supercoiled (see Important below). 2. Use the reaction mixture to transform a suitable E. coli ho