Introduction

The Ultimate™ ORF (Open Reading Frame) Clones are human and mouse clones designed to provide the maximum flexibility in all of your research applications. The Ultimate™ ORF Clones are provided in a Gateway® entry vector, pENTR™221, allowing you to rapidly and efficiently transfer the ORF of interest to any expression (Gateway® destination) vector and perform gene analysis in your system of choice.

Important

The amino acid sequence (coding sequence) of each Ultimate™ ORF Clone is guaranteed to match the corresponding GenBank amino acid sequence. The Ultimate™ ORF Clones are not guaranteed to exactly match the GenBank base pair sequences.

Features of Ultimate™ ORF Clones

The Ultimate™ ORF Clones are:

  • Full insert sequenced.   
    Each is sequenced for full-insert coverage and contains a Gap4 sequence quality of 40 or greater at each consensus base (representing < 1 error per 10,000 bases).
  • Supplied as a Gateway®-compatible entry clone Enables rapid and efficient transfer of the ORF into any expression (Gateway® destination) vector for performing gene analysis in bacterial, mammalian, yeast, or insect system of choice (see Note below).
  • Compatible for use with the Tag-On-Demand™ System
    Each clone contains a TAG stop codon, allowing expression of native or C-terminally-tagged recombinant protein in mammalian cells from the same expression construct using the Tag-On-Demand™ Suppressor Supernatant

Note
:  The pENTR™221 vector contains a Kozak consensus sequence upstream of the ATG to provide optimal expression of the ORF after recombination with any eukaryotic Gateway® destination vector of choice (e.g., mammalian, insect, yeast). The pENTR™221 vector does not contain a Shine-Dalgarno sequence (RBS) for optimal expression in a prokaryotic system (Shine & Dalgarno, 1975). To express the ORF in a prokaryotic system, you need to recombine the entry clone with an appropriate destination vector containing an N-terminal fusion tag. The N-terminal fusion tag in some destination vectors for prokaryotic expression includes a Shine-Dalgarno sequence (RBS) optimally spaced from an ATG initiation codon for proper translation initiation in E. coli.

You may use the following destination vectors available from Invitrogen that contain an N-terminal fusion tag for prokaryotic expression. 

VectorFusion TagCatalog no.
pDEST™15Glutathione-S-transferase11802-014
pDEST™176xHis11803-012
pBAD-DEST49pBAD-DEST49 His-Patch Thioredoxin12283-016

Note the expression of your protein with the N-terminal tag will increase the size of your recombinant protein. Be sure to account for any additional amino acids between the fusion tag and the start of your protein.

Features of pENTR™221

The Ultimate™ ORF Clones are provided in a Gateway® entry vector, pENTR™221. The pENTR™221 vector contains the following elements:

  • rrnB transcription termination sequences to prevent basal expression of the gene of interest in E. coli
  • attL1 and attL2 sites for site-specific recombination of the entry clone with a Gateway® destination vector (for more information, refer to the Gateway® Technology manual)
  • Kozak consensus sequence for efficient translation nitiation in eukaryotic systems (Kozak, 1987; Kozak, 1990; Kozak, 1991)
  • Kanamycin resistance gene for selection in E. coli
  • pUC origin for high-copy replication and maintenance of the plasmid in E. coli

The Gateway® Technology

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 using the Gateway® Technology, simply: 
  1. 1. Generate an expression clone by performing a LR recombination reaction between the Ultimate™ ORF entry clone and a Gateway® destination vector of choice. 

  2. Introduce your expression clone into the appropriate host (e.g. bacterial, mammalian, yeast, insect) and express your recombinant protein.
For more information about the Gateway® Technology, refer to the Gateway® Technology with Clonase™ II manual. This manual is available for downloading from our website (www.invitrogen.com) or by contacting Technical Support.

The Tag-On-Demand™ System

The Tag-On-Demand™ System uses adenoviral-based stop suppression technology to allow expression of an untagged (i.e. native) or C-terminally-tagged recombinant protein of interest in mammalian cells from a single expression vector. You may use one of the Tag-On-Demand™ Gateway® vectors (see page vi) to quickly and easily generate an expression construct or you may use one of the compatible expression vectors available from Invitrogen (see page 8). The System is based on stop suppression technology originally developed by RajBhandary and colleagues (Capone et al., 1985), and consists of two major components:

  • A mammalian expression vector into which the gene of interest will be cloned. This vector is in a configuration that is compatible with expression of a C-terminal recombinant fusion protein using the Tag-On-Demand™ System.
  • The Tag-On-Demand™ Suppressor Supernatant, a replication-incompetent adenovirus containing the human tRNAser suppressor. This tRNA suppressor is mutated to recognize the TAG (amber stop) codon and decode it as a serine. When added to mammalian cells, the Tag-On-Demand™ Suppressor Supernatant is transduced and provides a transient source of the tRNAser suppressor. When the expression construct encoding a gene of interest with a TAG stop codon is present in mammalian cells, the stop codon will be translated as serine, allowing translation to continue through the downstream reading frame (i.e. C-terminal tag). This results in transient production of a C-terminally-tagged fusion protein

For more information on the Tag-On-Demand™ System, see the Tag-On-Demand™ Suppressor Supernatant manual. This manual is available for downloading from our website at www.invitrogen.com or by contacting Technical Support .
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Methods - Using Ultimate™ ORF Clones

Introduction 

General guidelines for using the Ultimate™ ORF Clones are described in this section. 

To perform the LR recombination reaction with a Gateway® destination vector. To use the Tag-On-Demand™ System for expressing the Ultimate™ ORF Clone in a mammalian cell line of choice see below. 

Preparing Glycerol Stocks 

We recommend that you prepare a set of master stocks prior to using your Ultimate™ ORF clone. To prepare 5–10 glycerol master stocks for long-term storage: 

  1. Streak a small portion of the glycerol stock you received on an LB plate containing 50 μg/ml kanamycin. 
  2. Incubate the plate at 37° C overnight. 
  3. Isolate a single colony and inoculate into 5-10 ml of LB containing 50 µg/ml kanamycin. 
  4. Grow the culture to stationary phase (OD600 = 1–2). 
  5. Mix 0.8 ml of culture with 0.2 ml of sterile glycerol and transfer to a cryovial. 
  6. Store at –80° C. Use one master stock to create working stocks for regular use. 
     

Plasmid Preparation 

To isolate plasmid DNA, you need to grow a culture of T1-Phage Resistant E. coli containing your Ultimate™ ORF clone. Use LB medium containing 50 μg/ml kanamycin to select single colonies and to grow a culture. Use a culture volume appropriate for the amount of plasmid needed for your plasmid isolation method of choice. You will need ~150 ng of DNA for the LR recombination reaction. 

We recommend isolating plasmid DNA using a resin based method such as the PureLink™ HiPure Plasmid Miniprep Kit or the PureLink™ HiPure Plasmid Midiprep Kit. 

Recombination Site of pENTR™221

Below is the recombination site for pENTR™221. Features are indicated as follows:

  • The attL sites are properly spaced to indicate the correct reading frame for fusion of your gene of interest to an
    N-terminal tag following recombination with a destination vector
  • Shaded regions correspond to those DNA sequences transferred from the entry clone into the destination vector following recombination.
  • Underlined sequence corresponds to the Kozak consensus sequence (CACC).



Figure 1



Note

The last nucleotide T in the attL1 sequence is changed to an A in Ultimate™ ORF Clones to generate the Kozak consensus sequence. This change in the nucleotide sequence does not affect the efficiency of the subsequent LR recombination reaction.

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Performing the LR Reaction

Introduction

Each Ultimate™ ORF Clone is supplied as a Gateway®- compatible entry clone. To perform the LR recombination reaction, you will transfer the gene of interest into an attRcontaining destination vector to create an attB-containing expression clone. Genes in the Ultimate™ ORF entry clone are transferred to the destination vector backbone by mixing the DNAs with the Gateway® LR Clonase™ II enzyme mix. The resulting recombination reaction is then transformed into E. coli and the expression clone is selected. Recombination between the attR (destination vector) and attL (Ultimate™ ORF entry clone) sites replace the ccdB gene in the destination vector with the gene of interest and results in the formation of attB sites in the expression clone. A brief protocol to perform the LR recombination reaction is provided below. For more details on the LR recombination reaction, refer to the Gateway® Technology with Clonase™ II manual. This manual is available for downloading from our website at www.invitrogen.com or by contacting Technical Support .
For most applications, we recommend performing the LR recombination reaction using a:

  • Supercoiled attL-containing entry clone
  • Supercoiled attR-containing destination vector

Note: If your destination vector or entry clone is large (>10 kb), you may linearize either vector to increase recombinational efficiency. You may also relax the destination vector using topoisomerase I to increase efficiency. For details, refer to the Gateway® Technology with Clonase™ II manual. 

Destination Vectors 

A large selection of Gateway® destination vectors is available from Invitrogen to facilitate expression of your gene of interest in virtually any protein expression system. For more information about the vectors available, refer to our website (www.invitrogen.com) or contact Technical Support.


Destination Vectors Compatible with the Tag-On-Demand™ System

The following destination vectors available from Invitrogen are compatible for use with the Tag-On-Demand™ System: 

 

Vector Catalog no
pcDNA™6.2/V5-DESTK420-01*
pcDNA™3.2/V5-DEST 12489-019
pcDNA™-DEST40 12274-015
pcDNA™6.2/GFP-DEST K410-01*
pcDNA™-DEST47 12281-010
pEF5/FRT/V5-DEST V6020-20
pLenti4/V5-DEST K4980-00
pAd/CMV/V5-DEST K4930-00


*Includes the Tag-On-Demand™ Suppressor Supernatant

E. coli Host 

You may use any recA, endA E. coli strain including TOP10, DH5α™, DH10B™or equivalent for transformation. Do not transform the LR reaction mixture into E. coli strains that contain the F' episome (e.g. TOP10F'). These strains contain the ccdA gene and will prevent negative selection with the ccdB gene.


Materials Needed

You will need the following materials:

  • Purified plasmid DNA of your Ultimate™ ORF entry clone (50–150 ng/µl in TE, pH 8.0)
  • Destination vector of choice (150 ng/µl in TE, pH 8.0)
  • LR Clonase™ II enzyme mix (see Note below; keep at –20°C until immediately before use)
  • TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8, 1 mM EDTA)
  • 2 µg/µl proteinase K solution (supplied with the LR Clonase™ II enzyme mix; thaw and keep on ice until use)
  • Appropriate competent E. coli host and growth media
  • S.O.C. Medium
  • LB agar plates with the appropriate antibiotic to select for expression clones


LR Clonase™ II Enzyme Mix

Use LR Clonase™ II enzyme mix (Catalog no. 11791-020) to catalyze the LR recombination reaction. The LR Clonase™ II enzyme mix combines the proprietary enzyme formulation and 5X LR Clonase™ Reaction Buffer previously supplied as separate components in LR Clonase™ enzyme mix into an optimized single-tube format for easier set-up of the LR recombination reaction. Use the protocol below to perform the LR recombination reaction using LR Clonase™ II enzyme mix.

Note: You may perform the LR recombination reaction using LR Clonase™ enzyme mix, if desired. To use LR Clonase™ enzyme mix, follow the protocol provided with the product. Do not use the protocol for LR Clonase™ II enzyme mix as reaction conditions differ.

LR Recombination Reaction

  1. Add the following components to 1.5 ml microcentrifuge tubes at room temperature and mix.

    Component                                                 Sample                                      Negative Ctrl
    Entry clone (50–150 ng/rxn)                          1–7 µl                                           1–7 µl
    Destination Vector (150 ng/µl)                       1 µl                                               1 µl
    TE Buffer, pH 8.0                                            to 8 µl                                           to 10 µl


  2. Remove the LR Clonase™ II enzyme mix from –20°C and thaw on ice for 2 minutes.

  3. Vortex the LR Clonase™ II enzyme mix briefly twice (2 seconds each time).

  4. To each sample (except the negative control), add 2 µl of LR Clonase™ II enzyme mix. Mix by vortexing briefly twice (2 seconds each time). Return the LR Clonase™ II enzyme mix to –20°C immediately after use.

  5. Incubate reactions at 25°C for 1 hour.

    Note: One hour incubation generally yields a sufficient number of colonies for analysis; however, the length of the LR reaction can be extended up to 18 hours. For large plasmids (>10 kb), overnight incubation yields more colonies and is recommended.

  6. Add 1 µl of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37°C.

  7. Transform 1–2 µl of the reaction into a suitable E. coli host and select for expression clones (refer to the  Gateway® Technology with Clonase™ II manual on our website).

Note:
  You may store the LR reaction at –20°C for up to 1 week before transformation, if desired.

Expected Results

If you use E. coli cells with a transformation efficiency of  ≥ 1 x 108 cfu/µg, the LR reaction should yield > 5000 colonies if the entire reaction is transformed and plated.

Once you have obtained an expression clone, you are ready to express your recombinant protein. Refer to the manual for the destination vector you are using for guidelines and instructions to express your recombinant protein in the appropriate system. Manuals for all Gateway® destination vectors are available for downloading from our website (www.invitrogen.com) or by contacting Technical Support

Using the Tag-On-Demand™ System

Introduction 

The Ultimate™ ORF Clones are compatible for use in the Tag-On-Demand™ System to express native or C-terminally tagged recombinant protein in mammalian cells from the same expression construct using the Tag-On-Demand™ Suppressor Supernatant. This section provides brief guidelines to use the Tag-On-Demand™ Suppressor Supernatant. For detailed protocols, refer to the Tag-On-Demand™ Suppressor Supernatant manual available from www.invitrogen.com or Technical Support.

Materials Needed

  • Your mammalian cell line of choice (Reminder: Do not use 293 cells or any cell line that expresses E1)
  • Complete growth media for your cell line
  • Appropriately-sized tissue culture plates
  • Tag-On-Demand™ Suppressor Supernatant 
  • Purified plasmid DNA of the expression construct containing your gene of interest
  • Transfection reagent (see Method of Transfection, below)


Plasmid Preparation

Once you have generated your expression clone, you must isolate plasmid DNA for transfection. Plasmid DNA for transfection into eukaryotic cells must be clean and free from contamination with phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipid complexing, decreasing transfection efficiency. We recommend isolating plasmid DNA using a resin based method, such PureLink™ HiPure Plasmid Miniprep Kit or PureLink™ HiPure Plasmid Midiprep Kit or CsCl gradient centrifugation to isolate plasmid DNA.

Method of Transfection

Methods for transfection include calcium phosphate (Chen& Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner & Ringold, 1989) and electroporation (Chu et al., 1987; Shigekawa & Dower, 1988). Consult published literature or the supplier of your cell line for the recommended method of transfection and transfection reagent to use. For high-efficiency transfection in a broad range of mammalian cell lines, we recommend using Lipofectamine™ 2000 Reagent available from Invitrogen

 

Using the Tag-On-Demand™ System

For general guidelines and a detailed protocol, refer to the Tag-On-Demand™ Suppressor Supernatant manual available from www.invitrogen.com or Technical Support.

Two options exist to facilitate expression of C-terminally-tagged recombinant protein using the Tag-On-Demand™ System:

Option 1:   Add the Tag-On-Demand™ Suppressor Supernatant to cells, followed immediately by transfection with the expression construct containing your gene of interest. We recommend using this option to quickly screen for expression (or localization, if possible) of your recombinant protein or to screen for expression of a large number of genes. Refer to the Tag-On-Demand™ Suppressor Supernatant manual for guidelines and protocols to perform simultaneous transduction and transfection.

Option 2:   Generate a stable cell line expressing your gene of interest, then transduce cells with the Tag-On-Demand™ Suppressor Supernatant when you want to assay for C-terminally-tagged recombinant protein. After you have created your stable cell line, refer to the Tag-On-Demand™ Suppressor Supernatant manual to transduce cells with the Tag-On-Demand™ Suppressor Supernatant.

Important:

The Tag-On-Demand™ Suppressor Supernatant contains recombinant adenovirus that is deleted in the E1 region. The adenovirus is replication-incompetent in any mammalian cells that do not express the E1 proteins. When using the Tag-On-Demand™ System, do not perform expression studies in 293 cells or in any cell line that expresses the adenovirus E1 gene (Graham et al., 1977; Kozarsky & Wilson, 1993; Krougliak & Graham, 1995). Viral replication will occur in these cells, leading to rapid death of the target cell within 1–2 days after infection.

References

  1. Capone, J. P., Sharp, P. A., and RajBhandary, U. L. (1985) Amber, Ochre and Opal Suppressor tRNA Genes Derived from a Human Serine tRNA Gene. EMBO J. 4, 213-221

  2. Chen, C., and Okayama, H. (1987) High-Efficiency Transformation of Mammalian Cells by Plasmid DNA. Mol. Cell. Biol. 7, 2745-2752

  3. Chu, G., Hayakawa, H., and Berg, P. (1987) Electroporation for the Efficient Transfection of Mammalian Cells with DNA. Nucleic Acids Res. 15, 1311-1326

  4. Felgner, P. L., Holm, M., and Chan, H. (1989) Cationic Liposome Mediated Transfection. Proc. West. Pharmacol. Soc. 32, 115-121

  5. Felgner, P. L. a., and Ringold, G. M. (1989) Cationic Liposome-Mediated Transfection. Nature 337, 387-388

  6. Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977) Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5. J. Gen. Virol. 36, 59-74

  7. Kozak, M. (1987) An Analysis of 5´-Noncoding Sequences from 699 Vertebrate Messenger RNAs. Nucleic Acids Res. 15, 8125-8148

  8. Kozak, M. (1990) Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes. Proc. Natl. Acad. Sci. USA 87, 8301-8305

  9. Kozak, M. (1991) An Analysis of Vertebrate mRNA Sequences: Intimations of Translational Control. J. Cell Biology 115, 887-903

  10. Kozarsky, K. F., and Wilson, J. M. (1993) Gene Therapy: Adenovirus Vectors. Curr. Opin. Genet. Dev. 3, 499-503

  11. Krougliak, V., and Graham, F. L. (1995) Development of Cell Lines Capable of Complementing E1, E4, and Protein IX Defective Adenovirus Type 5 Mutants. Hum. Gene Ther. 6, 1575-1586

  12. Landy, A. (1989) Dynamic, Structural, and Regulatory Aspects of Lambda Sitespecific Recombination. Ann. Rev. Biochem. 58, 913-949

  13. Orosz, A., Boros, I., and Venetianer, P. (1991) Analysis of the Complex Transcription Termination Region of the Escherichia coli rrnB Gene. Eur. J. Biochem. 201, 653-659

  14. Shigekawa, K., and Dower, W. J. (1988) Electroporation of Eukaryotes and Prokaryotes: A General Approach to the Introduction of Macromolecules into Cells. BioTechniques 6, 742-751

  15. Shine, J., and Dalgarno, L. (1975) Terminal-Sequence Analysis of Bacterial Ribosomal RNA. Correlation Between the 3'-Terminal-Polypyrimidine Sequence of 16-S RNA and Translational Specificity of the Ribosome. Eur. J. Biochem. 57, 221-230

  16. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.-C., and Axel, R. (1977) Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells. Cell 11, 223-232
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