( ...)New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence ,,, Humanized Renilla GFP Reporter

New Vitality hrGFP expression vectors for monitoring gene expression and localizing proteins in vivo

New Mammalian Expression Vectors Employ Stable, High-Level Fluorescence Humanized Renilla GFP Reporter

Brenda Rogers Keith Chen Katherine Felts

Stratagene

Stratagene has added new vectors to the Vitality hrGFP mammalian expression vector product line ^Y. The new Vitality phrGFP-C and phrGFP-N1 protein fusion vectors, like the original Vitality pIRES-hrGFP-1a and pIRES-hrGFP-2a bicistronic expression vectors , are used for monitoring gene expression and protein localization. The Vitality vectors encode the humanized Renilla reniformis green fluorescent protein (hrGFP), a reporter protein known for its low cellular toxicity. Expression studies with the hrGFP reporter maintain a healthy cell population and ensure that results are biologically valid. Related products include the Vitality pFB-hrGFP retroviral vector and transduction-ready retroviral supernatants, useful controls for retroviral gene expression systems, and a variety of subcellular localization vectors. With this array of hrGFP-based products, expression of your gene of interest can be optimized for your specific applications. Here, we confirm the utility and versatility of Vitality vectors, illustrated in a variety of expression studies.

The green fluorescent protein (GFP) gene from the sea pansy Renilla reniformis has recently been isolated, adapted to use prevalent human codons, and developed as a reporter for mammalian expression vectors by Stratragene.¹ In contrast to the jellyfish Aequoria victoria enhanced GFP (EGFP), humanized Renilla GFP (hrGFP) has low cellular toxicity. This important attribute resolves problems that arise from the high cytotoxicity of EGFP, which triggers events that change the gene expression profile of cells and can confine experiments to the limited window of cell viability. In addition to maintaining cellular viability, hrGFP generates high-level fluorescence that can easily be detected in vivo by fluorescence microscopy or fluorescence activated cell-sorting (FACS) analysis.

Stratagenes Vitality vectors are specialized mammalian expression vectors that contain the hrGFP reporter. All Vitality vectors provide the following capabilities: generating stable cell lines expressing hrGFP as the reporter; detecting transfected cells via expression of hrGFP reporter; exchanging hygromycin, neomycin, and puromycin drug-resistance genes by Cre-Lox recombination; confirming transfection efficiency, and detecting fluorescence in vivo with the hrGFP reporter. Moreover, Vitality vectors are versatilethey are designed so that a gene of interest can be conveniently inserted in a variety of vectors for protein expression. Vitality vectors offer a variety of expression configurations that can be customized and optimized to best fit specific applications.

Bicistronic Expression Vectors

Fig.1

The pIRES-hrGFP-1a and pIRES-hrGFP-2a expression vectors (Figure 1) have a multiple cloning site upstream of three contiguous copies of either the FLAG or hemagglutin (HA) epitope tag sequence, respectively. In both vectors, duplicate stop codons follow the final epitope tag sequence to ensure termination of the inserted gene transcript and expression of an epitope-tagged native protein. The stop codons are followed by the coding region of an internal ribosome entry site (IRES) for reentry of the ribosome and protein production of the downstream hrGFP gene. In these bicistronic vectors, translation of two open reading frames from one mRNA allows expression of a gene of interest to be monitored at the single-cell level by virtue of expression of hrGFP on the same transcript. Expression of hrGFP can be detected visually, and the FLAG- or HA-tagged fusion product is easily detected by Western blot analysis.

We used a Vitality bicistronic vector to monitor expression of the luciferase gene, cloned into the multiple cloning site, and hrGFP. We transfected HeLa cells with the pIRES-hrGFP-1a vector carrying the luciferase gene. As can be seen by fluorescent microscopy, hrGFP expression is vivid (Figure 2). Western blot analysis clearly indicates expression of the luciferase protein (Figure 2).

Fig.2

New Expression Vectors Generate hrGFP Fusion Proteins

The newest Vitality vectors are the phrGFP-N1 and phrGFP-C protein fusion vectors (Figure 1). These vectors are powerful tools for localizing recombinant protein expression in cells. With the phrGFP-N1 vector, insertion of a gene of interest into the multiple cloning site results in expression of hrGFP fused to the N-terminus of the protein of interest. Inserts into the phrGFP-C vector are expressed as C-terminal fusions to hrGFP.

We tested the protein fusion vectors by fusing hrGFP to a series of genes that encode subcellularly localized proteins. In Figure 3, each fluorescent microscope photograph shows hrGFP expression localized to the organelle that corresponds to the appropriate fusion protein. These data confirm that the phrGFP-N1 and phrGFP-C protein fusion vectors provide convenient plasmid construction and high-level fluorescence of hrGFP fusion proteins within the cell. In addition, each of the four subcellular localization vectors shown in Figure 3 are available as stand alone products. These vectors allow real-time monitoring of the fate of peroxisomes, mitochondria, golgi and nuclei in both transient and stable applications.

Fig.3

Useful hrGFP-Based Controls for Retroviral Expression Systems

Fig.4

Along with mammalian expression vectors, the hrGFP gene is used in the pFB-hrGFP retroviral vector (Figure 4) as a reporter vector for determining the efficiency of retroviral transductions where expression cannot be determined directly. Transduction efficiency and gene expression of the pFB or pFB-neo retroviral vectors containing genes of interest can be confirmed indirectly, by conducting an additional transduction experiment of the reporter pFB-hrGFP in parallel. This vector is ideal for use with Stratagenes ViraPort retroviral expression system as a tool to verify transduction efficiency.⁸

The pFB-hrGFP retroviral reporter vector is available as plasmid DNA and as a transduction-ready, replication-incompetent, vesicular stomatitis virus (VSV)-G pseudotyped high-titer retroviral supernatant. In plasmid form, the pFB-hrGFP retroviral vector must be packaged into infectious virion particles.³ We recommend using Stratagenes Vpack vectors which consist of a set of 5 vectors that can be used with any MMLV-based retroviral vector to produce viral supernatants in transient triple-transfection experiments. The vectors include a gag-pol-expressing vector that is cotransfected with the retroviral expression vector together with one of a choice of 4 envelope (env)-expressing vectors. The choice of the env-expressing vector is based on the range of cell types the user wishes to transduce. With the pVPack vector system, all of the cis and trans elements required to produce infectious virus are separated onto three plasmids, with minimal or no sequence overlap between the plasmids, resulting in the generation of replication-incompetent virions. As an alternative strategy, Stratagene also offers a pFB-hrGFP transduction-ready supernatant that may be used to immediately deliver the hrGFP gene to cells, thereby eliminating the process of generating infectious virus particles. The supernatants are titered for ease in controlling the desired multiplicity of infection.

Fig.5

To test the performance of our pFB-hrGFP retroviral vector, we used this vector to transduce several widely used mammalian cell types. Figure 5 illustrates the high-intensity fluorescence that resulted upon delivery of hrGFP to human, rodent, and simian cells. To further demonstrate the high transduction efficiency of this retroviral vector, we analyzed the fluorescence profile of transduced cells by FACS. These data showed that ~100% of the cells were transduced with pFB-hrGFP and that the fluorescence from hrGFP expression was vivid (data not shown).

Conclusions

The Vitality hrGFP vectors, which contain the humanized green fluorescent protein from Renilla reniformis (hrGFP), are useful mammalian expression vectors for monitoring gene expression and localizing proteins. Now, the hrGFP gene is available in a variety of vector formats for choosing the appropriate configuration for specific gene expression studies. In a variety of experiments, we used the original Vitality pIRES-hrGFP-1a and pIRES-hrGFP-2a vectors to express epitope-tagged proteins. We used the new Vitality phrGFP-N1 and phrGFP-C vectors to express hrGFP-protein fusions that targeted specific subcellular locations. In further tests, we used the Vitality pFB-hrGFP retroviral control vector to transduce a variety of mammalian cells. These tests confirmed the functionality and utility of these vectors and the high-intensity fluorescence of hrGFP expression. Most importantly, the low toxicity of hrGFP expression ensures that use of Vitality vectors results in artifact-free transient and stable experiments.

REFERENCES

1. Felts K., Rogers, B., Chen, K., Ji , H., Sorge, J., and Vaillancourt, P. (2000) Strategies 13: 85-87.
2. Fieck, A., et. al. (1995) Nucleic Acids Res. 20: 1785-91.
3. Masibay, A.S., et. al. (1993) J. Biol. Chem. 268: 9908-9916.
4. Rizzuto, R., et. al. (1995) Curr. Biol. 5: 635-642.
5. Gould, SG., et.al.. (1997) J. Cell Biol., 105(6Pt2): 2923-2931.
6. Miller, A. D. (1990) Retrovirus Packaging Cells. In Human Gene Therapy, pp. 514. Mary Ann Liebert, Inc.,Publishers.
7. Felts, K, Bauer, J., and Vaillancourt, P. (1999) Strategies 12: 7477.
8. Rogers, B., Felts K., and Vaillancourt, P. (2000) Strategies 13: 97-99.

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